Cloning and uses of the genetic locus bcl-6

ABSTRACT

This invention provides an isolated vertebrate nucleic acid molecule the bcl-6 locus. This invention also provides an isolated human nucleic acid molecule of bcl-6 locus. This invention further provides a nucleic acid molecule comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of the nucleic acid molecule of bcl-6 locus. This invention provides an isolated vertebrate nucleic acid molecule of bcl-6 operatively linked to a promoter of RNA transcription. This invention provides a vector which comprises the nucleic acid molecule of bcl-6 locus. This invention provides a host vector system for the production of a polypeptide encoded by bcl-6 locus, which comprises the vector of bcl-6 locus in a suitable host. This invention provides a polypeptide encoded by the isolated vertebrate nucleic acid molecule of bcl-6 locus. This invention provides an antibody capable of binding to polypeptide encoded by bcl-6 locus. This invention provides an antagonist capable of blocking the expression of the polypeptide encoded by bcl-6. This invention provides an antisense molecule capable of hybridizing to the nucleic acid molecule of bcl-6. This invention provides an assay for non-Hodgkin&#39;s lymphoma, a method for screening putative therapeutic agents for treatment of non-Hodgkin&#39;s lymphoma and a method for diagnosing B-cell lymphoma in a subject. Finally, this invention provides a method of treating a subject with non-Hodgkin&#39;s lymphoma.

[0001] This application is a continuation-in-part of United Statesapplication Ser. No. 08/074,967, filed on Jun. 9, 1993, the contents ofwhich are hereby incorporated by reference.

[0002] The invention disclosed herein was made with Government supportunder NIH Grant Nos. CA-44029, CA-34775, CA-08748 and CA-37295 from theDepartment of Health and Human Services. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various references are referred towithin parenthesis. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of each Experimental Detail Section.

[0004] Non-random chromosomal abnormalities are found in up to 90% ofpatients with non-Hodgkin's lymphoma (NHL) and have been shown to playan important role in lymphomagenesis by activating proto-oncogenes (1).Some of these translocations, which are associated with specifichistologic subsets of NHL, have been characterized at the molecularlevel. In the t(8;14), t(8;22), and t(2;8) translocations associatedwith Burkitt Lymphoma, L₃-type acute lymphoblastic leukemia andAIDS-associated non-Hodgkin lymphoma (NHL), a known proto-oncogene,c-myc, was found juxtaposed to the immunoglobulin (Ig) loci (2,3). Inthe t(14;18) translocation, which is implicated in follicular-type NHL,molecular analysis of the sequences linked to the Ig locus led to theidentification of a novel proto-oncogene, bcl-2 (4-6). Thet(11;14)(q13;q32), mainly associated with “mantle zone”, lymphoma,appears to involve the juxtaposition of the Ig heavy-chain locus withthe bcl-1 locus, the site of the candidate proto-oncogene PRAD-1/cyclinD1 (7,8). These well characterized chromosome translocations areassociated, however, with only a fraction of NHL cases, while a numberof other recurrent translocations remain to be characterized for theirgenetic components.

[0005] One important example of such cytogenetic abnormalities isrepresented by various alterations affecting band 3q27. This region isinvolved in translocations with various chromosomal sites including, butnot limited, to those carrying the Ig heavy-(14q32) or light-(2p12,22q11) chain loci (9,10). Overall, 3q27 breakpoints are detectable in7-12% of B-cell NHL cases by cytogenetic analysis, with t(3;22)(q27;q11)being the most frequent type detectable in 4-5% of NHL (9). Theclinicopathologic relevance of 3q27 breakpoints is underscored by itsconsistent association with diffuse-type NHL, a frequent and clinicalaggressive subtype for which no specific molecular lesion has yet beenidentified (9).

[0006] The recurrence of 3q27 breakpoints in NHL has prompted a searchfor the corresponding proto-oncogene. This invention discloses thecloning of clustered 3q27 breakpoints from two NHL cases carryingt(3;14)(q27;q32) translocations and the identification of genomicrearrangements within the same breakpoint region in additional NHL casescarrying translocations involving 3q27. Within the same region, atranscriptional unit has been identified, which represents the candidateproto-oncogene (bcl-6) associated with 3q27 translocations in B-NHL.

SUMMARY OF THE INVENTION

[0007] This invention provides an isolated vertebrate nucleic acidmolecule of bcl-6 locus. This invention provides an isolated vertebrateDNA molecule of bcl-6 locus. This invention provides an isolatedvertebrate cDNA molecule of bcl-6. This invention provides an isolatedgenomic DNA molecule of bcl-6. This invention provides an isolatedvertebrate RNA molecule of bcl-6. This invention provides an isolatedhuman nucleic acid molecule of bcl-6 locus.

[0008] In addition, this invention provides a nucleic acid moleculecomprising a nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence included within the sequence ofthe nucleic acid molecule of bcl-6.

[0009] In addition, this invention provides an isolated vertebrate DNAmolecule of bcl-6 operatively linked to a promoter of RNA transcription.This invention provides a vector which comprises the isolated vertebrateDNA molecule of bcl-6.

[0010] In addition, this invention provides the above vector, whereinthe isolated nucleic acid molecule is linked to a plasmid.

[0011] In addition, this invention provides a host vector system for theproduction of a polypeptide encoded by bcl-6 locus, which comprises theabove vector in a suitable host.

[0012] In addition, this invention provides a method of producing apolypeptide encoded by bcl-6 locus, which comprises growing the abovehost vector system under suitable conditions permitting production ofthe polypeptide and recovering the polypeptide so produced.

[0013] In addition, this invention provides a polypeptide encoded by theisolated vertebrate nucleic acid molecule of bcl-6 locus. Further, thisinvention provides an antibody capable of binding to polypeptide encodedby bcl-6 locus.

[0014] In addition, this invention provides an antagonist capable ofblocking the expression of the polypeptide encoded by bcl-6.

[0015] In addition, this invention provides an antisense moleculecapable of hybridizing to the nucleic acid molecule of bcl-6.

[0016] In addition, this invention provides an assay for non-Hodgkin'slymphoma, a method for screening putative therapeutic agents fortreatment of non-Hodgkin's lymphoma and a method for diagnosing B-celllymphoma.

[0017] Finally, this invention provides a method of treating a subjectwith non-Hodgkin's lymphoma.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1: Immunoglobulin gene rearrangement analysis of KC1445 andSM1444 DNA. DNA extracted from the cell lines U937 (monocytic leukemia)and SK-N-MC (neuroblastoma) were used as controls for non-rearranged,germ-line Ig genes. In the left panel, the arrow on the left points tothe rearranged J_(H) fragment which does not contain C_(μ) sequences inKC1445 DNA, while the two arrows on the right point to the two distinctfragments containing J_(H) or C_(μ) sequences in SM1444 DNA.

[0019]FIG. 2: Molecular cloning of the chromosomal breakpoints from twoNHL cases with t(3;14). Illustrated are the maps of two representativephage clones spanning the breakpoint regions in case SM1444 (SM-71) andKC1445 (KC-51). Chromosome 14 portions of the phage inserts areindicated by a solid line with hatched and black boxes representingswitch sequences and C_(μ) exons, respectively. Vertical arrows point tothe junctions of chromosome 3 and 14 sequences. The probes used forSouthern (FIG. 4) and Northern (FIG. 5) analysis are illustrated belowthe SM-71 map. Restriction enzyme sites are indicated as: B=BamHI;H=HindIII; R=EcoRI; G=BblII; S=sacI.

[0020]FIG. 3: Localization of phage SM-71 sequences to chromosomes 3 and14 by fluorescence in situ hybridization. Consistent hybridizationsignals at 3q27 (arrow in panel A) and 14q32 (arrow in panel B)demonstrated that the insert is derived from the translocation junction.

[0021] FIGS. 4A-4C: Southern blot hybridization analysis of bcl-6rearrangements in NHL carrying 3q27 breakpoints. The probes used areillustrated in FIG. 2. U937 and SK-N-MC DNAs are used as germ-linecontrols since their hybridization pattern was identical to the oneobserved in a panel of 19 control DNAs tested. The detected cytogeneticabnormalities affecting 3q27 in each case are: KC1445: t(3;14)(q27;q32);SM1444: t(3;14)(q27;q32); TF1403: t(3;14)(q27;q32); LD1411:t(3,14)(q27;q32); EM352 t(3;22)(q27;q); CF755 t(3;12)(q27;q11)SO955:der(3)t(3;5)(q27;q31).

[0022]FIG. 5: Identification of the bcl-6 transcriptional unit. 15 μg oftotal RNA isolated from the indicated human cell lines was analyzed byNorthern blot hybridization using the Sac 4.0 probe (see FIG. 2).CB33:EBV-immortalized human B lymphoblastoid cell line; HeLa: humancervical carcinoma cell line; Daudi: human Burkitt lymphoma cell line;Hut78: human T-cell leukemia cell line. Hybridization of the same filterto a mouse GAPDH probe is shown as control for RNA amount loaded in eachlane. The faint band comigrating with 28S RNA in all the lanes may bethe result of cross-hybridization with ribosomal RNA sequences.

[0023]FIG. 6: Map of normal human BCL-6 locus. A recombinant genomic DNAlibrary derived from normal placenta DNA was obtained from STRATAGENEInc and screened by plaque hybridization using the Sac 4.0 probe. Threerecombinant phages were obtained (φ 1-3 in the figure) whose insertshave been mapped and shown to overlap on approximately 30 kilobases ofgenomic DNA representing the BCL-6 locus. These sequences containingbcl-6 exons since they hybridize to the cDNA probe. The precise positionof the exons has only been approximately determined and is schematicallyindicated in the figure. The position of the breakpoints observed invarious lymphoma cases is also indicated.

[0024]FIG. 7: pSac 40 plasmid construction.

[0025]FIG. 8: pGB31 and pGB3s plasmid construction.

[0026] FIGS. 9A-9D: cDNA and Amino Acid Sequences of BCL-6 (SEQ ID NOs.1 and 2). The Sac 4.0 probe was used to screen a recombinant phage cDNAlibrary constructed from Bjab B cell lymphoma line RNA. A 4.0 kilobasecDNA was isolated and its nucleotide sequence was determined. Itcontains a long open reading frame potentially coding for 706 amino acidprotein which contains five zinc-finger domains (underlined in thefigure; C and H residues which identify the C2H2-type zinc-fingerstructure are indicated in bold).

[0027] FIGS. 10A-10B: Structure of BCL-6 cDNA and sequence of itspredicted protein product. FIG. 10A: Schematic representation of thefull-length BCL-6 cDNA clone showing the relative position of the openreading frame (box) with 5′ and 3′ untranslated sequences (linesflanking the box). The approximate positions of the zinc-finger motifs(Zn++) and the NH₂-terminal homology (shaded area) with other proteinsare also indicated. FIG. 10B: The predicted amino acid sequence of theBCL-6 protein. The residues corresponding to the six zinc-finger motifs(H-C links). The GenBank Accession number for BCL-6 cDNA and amino acidsequences is U00115.

[0028]FIG. 11: Homology of the NH₂-terminal region of BCL-6 to otherKrüppel zinc-finger proteins, viral (VA55R), or cellular non-zinc-finger(kelch) proteins. Black background indicates identical residues foundfour or more times at a given position; grey indicates conservedresidues that appear in at least four sequences at a given position.Conserved amino acid substitutions are defined according to scheme (P,A, G, S, T), (Q, N, E, D), (H, K, R), (L, I, V, M), and (F<Y<W)Numbering is with respect to the methionine initiation codon of eachgene.

[0029]FIG. 12: Exon-intron organization of the BCL-6 gene and mapping ofbreakpoints detected in DLCL. Coding and non-coding exons arerepresented by filled and empty boxes, respectively. The position andsize of each exon are approximate and have been determined by thepattern of hybridization of various cDNA probes as well as by thepresene of shared restriction sites in the genomic and cDNA. Theputative first, second and third exons have been sequenced in theportions overlapping the cloned cDNA sequences. The transcriptioninitiation site has not been mapped (shaded box on 5′ side of firstexon). Patient codes (e.e. NC11, 891546 etc.) are grouped according tothe rearranged patterns displayed by tumor samples. Arrows indicate thebreakpoint position for each sample as determined by restrictionenzyme/hybridization analysis. For samples KC1445 and SM1444, thebreakpoints have been cloned and the precise positions are known.Restriction sites marked by asterisks have been only partially mappedwithin the BCL-6 locus. Restriction enzyme symbols are: S, Sac I; B, BamHI; X, Xba I; H, Hind III, R, Eco RI; G, Bgl II; P, Pst I; Sc, Sca I;St, Stu I; Rs, Rsa I. Tumor samples were collected and analyzed forhistopathology at Memorial Sloan-Kettering Cancer Center or at ColumbiaUniversity.

[0030] FIGS. 13A-13B: Rearrangements of the BCL-6 gene in diffuselarge-cell lymphomas (DLCL). Genomic DNA extracted from tumor biopsiesof DLCL cases and from normal lymphocytes (lane N) was digested with theindicated restriction enzymes and analyzed by Southern blothybridization using the Sac 4.0 probe. Abnormal restriction fragmentsare indicated by the arrows.

[0031] FIGS. 14A-14C: Analysis of BCL-6 rearrangements in AIDS-NHL(FIGS. 14A-14C). DNAs were digested with BamHI (FIG. 14A) or XbaI (FIGS.14B and 14C) and hybridized to probes Sac4.0 (FIGS. 14A and 14B) orSac0.8 (FIG. 14C). The BCL-6 germline bands detected by BamHI (11.4 Kb)and XbaI (14 Kb) are indicated. U937 was used as a BCL-6 germlinecontrol. Among the cases shown, rearrangements were detected in casesDK782, DK827, and DS16, represented by AIDS-DLCL.

[0032]FIG. 15: Restriction map of the germline BCL-6 locus. Exon-intronorganization of the BCL-6 gene. Coding and noncoding exons arerepresented by filled and empty boxes, respectively. The transcriptioninitiation site has not been mapped (shaded box on 5′ side of firstexon). The breakpoints detected in AIDS-NHL are indicated by arrows.Restriction enzyme symbols are: S, SacI; B, BamHI; X, XbaI, R, EcoRI.RE, restriction enzyme.

[0033] FIGS. 16A-16C: Analysis of EBV infection (FIG. 16A), c-MYCrearrangements (FIG. 16B), and p53 mutations (FIG. 16C) in AIDS-NHL.FIG. 16A: Analysis of EBV termini heterogeneity in AIDS-NHL. DNAs weredigested with BamHI and subjected to Southern hybridization using a DNAprobe specific for the fused termini of the EBV genome. U937, amonocytic leukemia cell line, is used as a negative control. Alymphoblastoid cell line derived by EBV infection of normal polyclonal Bcells (NC2) is used as control for polymorphic EBV termini.Representative samples of AIDS-NHL, both positive (DK3794, DK4338,DK2814, DK3973) and negative (DK3479), are shown. FIG. 16B: Southernblot analysis of c-MYC rearrangements in AIDS-NHL. Genomic DNAs from thecases shown was digested with HindIII and probed with clone MC413RC⁴¹,representative of c-MYC exon 3. A lymphoblastoid cell line (NC2) wasused as control for c-MYC germline configuration. Among the cases shown,two cases of AIDS-DLCL (DK3537 and DK1446) display a c-MYCrearrangement. FIG. 16C: Analysis by PCR-SSCP of the p53 gene inAIDS-NHL. Representative examples are shown for p53 exon 5. Samples werescored as abnormal when differing from the normal control (N). A sampleknown to harbor a p53 mutation was used as positive control (POS). Amongthe cases shown, DK1171, a case of AIDS-SNCCL, shows a p53 mutationwhich was further characterized by direct sequencing of the PCR product.

[0034] FIGS. 17A-17B: Southern blot analysis of the BCL-6 geneconfiguration in diffuse large cell lymphomas. Genomic DNA-extractedfrom tumor biopsies was digested with the indicated restrictionendonucleases and hybridized using the Sac4.0 probe (19). Rearrangedfragments are indicated by the arrows. N=normal control DNA obtainedfrom human lymphocytes.

[0035] FIGS. 18A-18B: FIG. 18A: Freedom from progression in BCL-6rearranged cases (open circles, top curve) compared to BCL-6 germlinecases (closed circles, bottom curve)(P=0.007). FIG. 18B: Overallsurvival from time of diagnosis for BCL-6 rearranged CLLC (open circle,top curve), compared to BCL-6 germline, BCL-2 germline DLLC (darktriangles, middle curve), and BCL-2 rearranged DLLC (dark boxes, bottomcurve) (P=0.02).

DETAILED DESCRIPTION OF THE INVENTION

[0036] The following standard abbreviations are used throughout thespecification to indicate specific nucleotides:

[0037] C=cytosine A=adenosine

[0038] T=thymidine G=guanosine

[0039] This invention provides an isolated vertebrate nucleic acidmolecule of the bcl-6 locus. As used herein, bcl-6 locus means thebreakpoint cluster region in B-cell lymphomas. The bcl-6 locus is of 30kilobase in length containing at least a bcl-6 gene which codes for aprotein. Therefore, the bcl-6 locus contains both the 5′ and 3′ flankingregion of the coding sequences of the bcl-6 gene.

[0040] In an embodiment, the isolated, vertebrate nucleic acid moleculeof bcl-6 locus is DNA. In another embodiment, the isolated, vertebratenucleic acid of the bcl-6 locus is cDNA. In a further embodiment, theisolated, vertebrate nucleic acid is genomic DNA. In a still furtherembodiment, the isolated, vertebrate nucleic acid molecule is RNA.

[0041] This invention provides an isolated, human nucleic acid moleculecomprising the bcl-6 locus.

[0042] The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

[0043] Moreover, the isolated vertebrate nucleic acid molecules areuseful for the development of probes to study B cell lymphomas.

[0044] This invention provides a nucleic acid molecule comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of the bcl-6locus. In an embodiment, this molecule is DNA. In another embodiment,the molecule is RNA.

[0045] As used herein, the phrase “specifically hybridizing” means theability of a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

[0046] The above nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence of bcl-6 locus maybe used as a probe for bcl-6 sequences. Nucleic acid probe technology iswell known to those skilled in the art who will readily appreciate thatsuch probes may vary greatly in length and may be labeled with adetectable label, such as a radioisotope or fluorescent dye, tofacilitate detection of the probe. DNA probe molecules may be producedby insertion of a DNA molecule having the full-length or a fragment ofthe bcl-6 locus into suitable vectors, such as plasmids orbacteriophages, followed by transforming into suitable bacterial hostcells, replication in the transformed bacterial host cells andharvesting of the DNA probes, using methods well known in the art.Alternatively, probes may be generated chemically from DNA synthesizers.

[0047] RNA probes may be generated by inserting the full length or afragment of the bcl-6 locus downstream of a bacteriophage promoter suchas T3, T7 or SP6. Large amounts of RNA probe may be produced byincubating the labeled nucleotides with a linearized bcl-6 or itsfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

[0048] This invention provides an cDNA molecule of bcl-6 locusoperatively linked to a promoter of RNA transcription.

[0049] This invention provides a vector which comprises the nucleic acidmolecule of bcl-6 locus. This invention provides the above vector,wherein the isolated nucleic acid molecule is linked to a plasmid.

[0050] This invention further provides isolated cDNA molecule of thebcl-6 locus operatively linked to a promoter of RNA transcription.Various vectors including plasmid vectors, cosmid vectors, bacteriophagevectors and other viruses are well known to ordinary skilledpractitioners.

[0051] As an example to obtain these vectors, insert and vector DNA canboth be exposed to a restriction enzyme to create complementary ends onboth molecules which base pair with each other and are then ligatedtogether with DNA ligase. Alternatively, linkers can be ligated to theinsert DNA which correspond to a restriction site in the vector DNA,which is then digested with the restriction enzyme which cuts at thatsite. Other means are also available and known to an ordinary skilledpractitioner.

[0052] In an embodiment, a partial cDNA molecule of the bcl-6 locus islinked to pGEM-7zf(−) and the resulting plasmid is designated as pGB31(FIG. 8). Plasmid, pGB31 was deposited on June 3, 1993 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganism for thePurposes of Patent Procedure. Plasmid, pGB31 was accorded with ATCCAccession Number 75476.

[0053] In an another embodiment, a partial cDNA molecule of the bcl-6locus is linked to pGEM-7zf(−) and the resulting plasmid is designatedas pGB3s (FIG. 8). Plasmid, pGB3s was deposited on June 3, 1993 with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganism forthe Purposes of Patent Procedure. Plasmid, pGB3s was accorded with ATCCAccession Number 75477.

[0054] This invention provides a host vector system for the productionof a polypeptide encoded by bcl-6 locus, which comprises the abovevector in a suitable host.

[0055] This invention provides the above host vector system, wherein thesuitable host is a bacterial cell, insect cell, or animal cell.

[0056] Regulatory elements required for expression include promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes a promoter such as the lac promoter and for transcriptioninitiation the Shine-Dalgarno sequence and the start codon AUG.Similarly, a eukaryotic expression vector includes a heterologous orhomologous promoter for RNA polymerase II, a downstream polyadenylationsignal, the start codon AUG, and a termination codon for detachment ofthe ribosome. Such vectors may be obtained commercially or assembledfrom the sequences described by methods well-known in the art, forexample the methods described above for constructing vectors in general.Expression vectors are useful to produce cells that express thepolypeptide encoded by the bcl-6 locus.

[0057] This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E. coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

[0058] This invention provides a method of producing a polypeptideencoded by bcl-6 locus, which comprises growing the above host vectorsystem under suitable conditions permitting production of thepolypeptide and recovering the polypeptide so produced.

[0059] This invention provides a polypeptide encoded by the isolatedvertebrate nucleic acid molecule of bcl-6 locus.

[0060] This invention provides an antibody capable of binding topolypeptide encoded by bcl-6 locus. In an embodiment, the antibody ismonoclonal.

[0061] This invention provides a method to select specific regions onthe polypeptide encoded by the bcl-6 locus to generate antibodies. Theprotein sequence may be determined from the cDNA sequence. Amino acidsequences may be analyzed by methods well known to those skilled in theart to determine whether they produce hydrophobic or hydrophilic regionsin the proteins which they build. In the case of cell membrane proteins,hydrophobic regions are well known to form the part of the protein thatis inserted into the lipid bilayer of the cell membrane, whilehydrophilic regions are located on the cell surface, in an aqueousenvironment. Usually, the hydrophilic regions will be more immunogenicthan the hydrophobic regions. Therefore the hydrophilic amino acidsequences may be selected and used to generate antibodies specific topolypeptide encoded by the bcl-6 locus. The selected peptides may beprepared using commercially available machines. As an alternative, DNA,such as a cDNA or a fragment thereof, may be cloned and expressed andthe resulting polypeptide recovered and used as an immunogen.

[0062] Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of polypeptide encoded bythe bcl-6 locus in living animals, in humans, or in biological tissuesor fluids isolated from animals or humans.

[0063] The antibody may be labelled with a detectable marker, includingbut not limited to: a radioactive label, or a calorimetric, luminescent,or fluorescent marker, or gold. Radioactive labels include but are notlimited to: ³H, ¹⁴C, ³²P, ³³P; ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁹Co, ⁵⁹Co, ⁵⁹Fe, ⁹⁰Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Fluorescent markers include but are not limitedto: fluorescein, rhodamine and auramine. Methods of producing thepolyclonal or monoclonal antibody are known to one of ordinary skill inthe art.

[0064] Further, the antibody complex may be detected by a secondantibody which may be linked to an enzyme, such as alkaline phosphataseor horseradish peroxidase. Other enzymes which may be employed are wellknown to one of ordinary skill in the art.

[0065] This invention provides for the isolated nucleic acid molecule ofbcl-6 that is labelled with a detectable marker. The detectable markermay be a radioactive label, a calorimetric, luminescent, or afluorescent marker. Other detectable markers are known to those skilledin the art as hereinabove described.

[0066] This invention provides an antagonist capable of blocking theexpression of the polypeptide encoded by the isolated nucleic acidmolecule of bcl-6. The antagonist may be a triplex oligonucleotidecapable of hybridizing to nucleic acid molecule bcl-6.

[0067] This invention provides an antisense molecule capable ofhybridizing to the nucleic acid molecule bcl-6. The antisense moleculemay be DNA or RNA.

[0068] This invention provides a triplex oligonucleotide capable ofhybridizing with a double stranded DNA molecule bcl-6.

[0069] The antisense molecule may be DNA or RNA or variants thereof(i.e. DNA with a protein backbone). The present invention extends to thepreparation of antisense nucleotides and ribozymes that may be used tointerfere with the expression of the receptor recognition proteins atthe translation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

[0070] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule. In thecell, they hybridize to that mRNA, forming a double stranded molecule.The cell does not translate an mRNA in this double-stranded form.Therefore, antisense nucleic acids interfere with the expression of mRNAinto protein. Oligomers of about fifteen nucleotides and molecules thathybridize to the AUG initiation codon will be particularly efficient,since they are easy to synthesize and are likely to pose fewer problemsthan larger molecules upon introduction to cells.

[0071] This invention provides a transgenic nonhuman mammal whichcomprises the isolated nucleic acid molecule bcl-6 introduced into themammal at an embryonic stage.

[0072] This invention provides an assay for non-Hodgkin's lymphoma,comprising (a) incubating a sample of suitable body fluid for a subjectwith a monoclonal antibody reactive with non-Hodgkin's lymphoma cells toa solid support, (b) removing unbound body fluid from the support, and(c) determining the level of antigen activity exhibited by the boundbody fluid to the support.

[0073] The suitable bodily fluid sample is any bodily fluid sample whichwould contain non-hodgkin lymphoma cells or fragments thereof. Asuitable bodily fluid includes, but is not limited to, serum, plasma,cerebrospinal fluid, and urine. In the preferred embodiment, thesuitable bodily fluid sample is serum or plasma. In addition, the bodyfluid sample may cells from bone marrow, or a supernate from a cellculture. Methods of obtaining a suitable bodily fluid sample from asubject are known to those skilled in the art.

[0074] This invention provides a method for screening putativetherapeutic agents for treatment of non-Hodgkin's lymphoma, whichcomprises determining in a first sample from a subject withnon-Hodgkin's lymphoma the presence of the isolated nucleic acidmolecule bcl-6, administering to the subject a therapeutic amount of theagent such that the agent is contacted with the cell associated with thecondition, determining after a suitable period the amount of theisolated nucleic acid molecule in a sample from the treated subject, andcomparing the amount of isolated nucleic acid molecule determined in thefirst sample with the amount determined in the sample from the treatedsubject, a difference indicating the effectiveness of the agent, therebyscreening putative therapeutic agents for treatment of non-Hodgkin'slymphoma.

[0075] Further, this invention provides an assay system that is employedto identify drugs or other molecules capable of binding to the nucleicacid molecule bcl-6 or proteins, either in the cytoplasm or in thenucleus, thereby inhibiting or potentiating transcriptional activity.Such assay would be useful in the development of drugs that would bespecific against particular cellular activity, or that would potentiatesuch activity, in time or in level of activity.

[0076] The above described probes are also useful for in-situhybridization or in order to locate tissues which express this gene, orfor other hybridization assays for the presence of this gene or its mRNAin various biological tissues.

[0077] The in-situ hybridization technique using the labelled nucleicacid molecule bcl-6 is well known in the art. Essentially, tissuesections are incubated with the labelled nucleic acid molecule to allowthe hybridization to occur. The molecule will carry a marker for thedetection because it is “labelled”, the amount of the hybrid will bedetermined based on the detection of the amount of the marker. Further,immunohistochemical protocols may be employed which are known to thoseskilled in the art.

[0078] This invention provides a method of diagnosing diffuse-typeB-cell lymphoma in a subject which comprises detecting in a sample fromthe subject nucleic acid molecule of bcl-6 locus.

[0079] This invention provides a method for diagnosing B-cell lymphomain a subject comprising: (a) obtaining DNA sample from the subject; (b)cleave the DNA sample into fragments; (c) separating the DNA fragmentsby size fractionation; (d) hybridizing the DNA fragments with a nucleicacid molecule comprising a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence includedwithin the sequence of the nucleic acid molecule of the bcl-6 locus todetect the DNA fragment containing the bcl-6 sequence; and (e) comparingthe detected DNA fragment from step (d) with the DNA fragment from aknown normal subject, the difference in size of the fragments indicatingthe occurrence of B-cell lymphoma in the subject. In a preferredembodiment, the above diagnostic method is for diffuse-type B-celllymphomas.

[0080] A person of ordinary skill in the art will be able to obtainappropriate DNA sample for diagnosing B-cell lymphoma in a subject. TheDNA sample obtained by the above described method may be cleaved byrestriction enzyme. The uses of restriction enzymes to cleave DNA andthe conditions to perform such cleavage are well-known in the art.

[0081] In an embodiment, the size fractionation in step (c) of theabove-described method is effected by a polyacrylamide gel. In anotherembodiment, the size fractionation is effected by an agarose gel.

[0082] This invention also provides the above-described diagnosis methodwherein step the nucleic acid molecule in step (d) is labeled with adetectable marker. The detectable marker includes but is not limited toa radiolabelled molecule, a fluorescent molecule, an enzyme, or aligand.

[0083] In a preferred embodiment, the above-described diagnosis methodfurther comprises transferring the DNA fragments into a solid matrixbefore the hybridization step (d). One example of such solid matrix isnitrocellulose paper.

[0084] As an example for the above-described diagnosis method is shownin FIGS. 4A-4C where different NHL sample are analyzed. More lymphomacases and their breakpoints are shown in FIG. 6.

[0085] This invention also provides a method for diagnosing B-celllymphoma in a subject comprising: (a) obtaining RNA sample from thesubject; (b) separating the RNA sample into different species by sizefractionation; (c) hybridizing the RNA species with a nucleic acidmolecule comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of the nucleic acid molecule of the bcl-6 locus to detect theRNA species containing the bcl-6 sequence; and (d) comparing the RNAspecies obtained from (c) with the RNA species from a known normalsubject, the difference in size of the species indicating the occurrenceof B-cell lymphoma in the subject.

[0086] In an embodiment, the size fractionation in step (b) is effectedby a polyacrylamide or agarose gel.

[0087] This invention also provides the above-described method where instep (c), the nucleic acid molecule is labeled with a detectable marker.The detectable marker includes but is not limited to a radiolabelledmolecule, a fluorescent molecule, an enzyme, or a ligand.

[0088] This invention also provides the above-method further comprisestransferring the RNA species into a solid matrix before step (c).

[0089] This invention also provides various uses of bcl-6 locus/gene anits derivatives. This invention further provides a method for diagnosisof B cell lymphoma and/or diffuse-type B cell lymphoma using bcl-6 DNAprobes or synthetic oligonucleotide primers derived from bcl-6 sequencesto detect bcl-6 rearrangements/mutations by Southern blotting PCR orother DNA based techniques.

[0090] This invention also provides a method of diagnosis of B celllymphoma and/or diffuse-type B cell lymphoma using bcl-6 DNA probes orsynthetic oligonucleotide primers derived from bcl-6 sequences to detectabnormal bcl-6 RNA species by Northern blotting, PCR or other RNA-basedtechniques.

[0091] This invention further provides a method of diagnosis of B celllymphoma and/or diffuse-type B cell lymphoma using antiserum ormonoclonal antibodies directed against the bcl-6 protein product(s).

[0092] This invention provides a method of treating a subject withnon-Hodgkin's lymphoma comprising administering an effective amount ofthe antisense molecule of the nucleic acid molecule bcl-6 operativelylinked to a suitable regulatory element coupled with a therapeutic DNAinto a tumor cell of a subject, thereby treating the subject withnon-Hodgkin's lymphoma.

[0093] This invention provides a method of treating a subject withnon-Hodgkin's lymphoma, comprising administering an effective amount ofthe antagonist capable of blocking the expression of the polypeptideencoded by the isolated nucleic acid molecule of bcl-6, and a suitableacceptable carrier, thereby treating the subject with non-Hodgkin'slymphoma.

[0094] Further, as is known to those of ordinary skill in the arteffective amounts vary with the type of therapeutic agent. It is knownto those of ordinary skill in the art how to determine an effectiveamount of a suitable therapeutic agent.

[0095] The preparation of therapeutic compositions which containpolypeptides, analogs or active fragments as active ingredients is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions, however, solidforms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.The active therapeutic ingredient is often mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents which enhance the effectiveness of the active ingredient.

[0096] A polypeptide, analog or active fragment can be formulated intothe therapeutic composition as neutralized pharmaceutically acceptablesalt forms. Pharmaceutically acceptable salts include the acid additionsalts (formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0097] The subjects contained herein may be a mammal, or morespecifically a human, horse, pig, rabbit, dog, monkey, or rodent. In thepreferred embodiment the subject is a human.

[0098] The compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.

[0099] Suitable regimes for initial administration and booster shots arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration.

[0100] As used herein administration means a method of administering toa subject. Such methods are well known to those skilled in the art andinclude, but are not limited to, administration topically, parenterally,orally, intravenously, intramuscularly, subcutaneously or by aerosol.Administration of the agent may be effected continuously orintermittently such that the therapeutic agent in the patient iseffective to treat a subject with non-hodgkin's lymphoma.

[0101] Finally, this invention provides a therapy of B cell lymphomaand/or diffuse-type B cell lymphoma using anti bcl-6 reagents includingspecific antisense sequences and compounds interfering with bcl-6functions.

[0102] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Detail Section I Materials and Methods

[0103] DNA Extraction and Southern Blot Analysis. Total genomic DNA waspurified from frozen tumor biopsies by cell lysis, proteinase Kdigestion, “salting-out” purification and ethanol precipitation aspreviously described (11). Southern blot hybridization analysis wasperformed in 50% formamide, 3× SSC, 10× dextran sulphate, 5× Denhardt'ssolution, 0.5% SDS at 37° C. for 16 hrs. Filters were washed in 0.2×SSC, 0.5% SDS at 60° C. for 2 hrs. DNA probes were ³²P-labelled by therandom priming method (12).

[0104] DNA Probes. The following probes were used for Southern blotanalysis of Ig gene rearrangements: i) (J_(H)) probe: 6.6 kbBamHi/HindIII fragment from the human Ig heavy-chain (Ig_(H)) locus(13); ii) (C_(μ)) probe: 1.3 kb EcoRI fragment containing the first twoexons of human C_(μ) (13).

[0105] Genomic Cloning. Genomic libraries from NHL cases SM1444 andKC1445 were constructed by partial Sau 3A restriction digestion ofgenomic DNA and ligation of gel-purified 15-20 kb fractions intoLambdaGem-11 phage vector (Promega). Library screening was performed byplaque-hybridization using the C_(μ) probe.

[0106] Fluorescence in situ Hybridization Analysis (FISH). Phage DNA waslabelled with biotin-14-dATP by nick translation and hybridized tometaphase spreads from normal human lymphocytes as described (14). Tovisualize the hybridization signal and the corresponding bandssequentially under the microscope, the slides were stained andcounterstained with propidium iodide and 4′6′-diamideno-2-phenylindole(DAPI), respectively.

[0107] Northern Blot Hybridization Analysis. RNAs from several humancell lines were extracted by the guanidine-isothiocyanate method (15).For Northern blot analysis, RNA samples were electrophoresed through0.9% agarose-2.2M formaldehyde gels and then transferred tonitrocellulose filters. Hybridization and washing were performed asdescribed for Southern blot analysis.

Experimental Results

[0108] DNA was extracted from tumor tissue of two cases (SM1444 andKC1445) of IgM-producing, diffuse-type B-cell NHL carrying thet(3;14)(q27;q32) translocation. Since the involvement of the Ig_(H)locus was suspected based on the 14q32 breakpoint, SM1444 and KC1445DNAs were first analyzed by Southern blot hybridization usingcombinations of enzymes and probes specific for the J_(H) and C_(μ)regions of the Ig_(H) locus (13). In both cases, digestion by BamHIshowed rearranged fragments containing J_(H) sequences (FIG. 1).Subsequent hybridizations to the C_(μ) probe showed, in each case, thatone rearranged fragment containing J_(H) sequences was not linked toC_(μ) sequences (see failure of the C_(μ) probe to hybridize to the samerearranged BamHI fragment detected by J_(H) (FIG. 1) as would beexpected for the physiologically rearranged Ig_(H) allele in IgMproducing cells. In addition, in both cases, digestion with HindIII andhybridization with C_(μ) detected a rearranged fragment, a findinginconsistent with either germ-line or physiologically rearranged Ig_(H)genes, since both HindIII sites flanking C_(μ) sequences are notinvolved in V-D-J arrangements (13). The observed pattern is, however,consistent with chromosomal breakpoints located within C_(μ) switchsequences, as previously observed in several cases of chromosomaltranslocations involving the Ig_(H) locus (2,16-18).

[0109] Based on this analysis, the C_(μ) containing fragments from eachcase were cloned by screening genomic libraries constructed from SM1444and KC1445 DNAs using the C_(μ) probe. Restriction mapping andhybridization analysis of several phage clones led to the identificationof recombinant phages from each library which contained C_(μ) sequenceslinked to sequences unrelated to the Ig_(H) locus (see FIG. 2 for mapsof representative phage clones). The Ig portions of the phage insertsoverlapped along the C_(μ) region extending 5′ into the switch regionwhere alignment with the restriction map of the normal Ig heavy-chainlocus was lost. The location of the breakpoint within C_(μ) switchsequences was confirmed for case SM1444 by DNA sequence analysis of thebreakpoint junction of phage SM-71, which revealed the presence of therepeated motifs typical of the Ig_(H) switch regions on the chromosome14 side (19). The Ig-unrelated portions of phage SM-71 and KC-51 alsooverlapped with each other in their restriction maps, suggesting thatthey were derived from the same genomic region. This notion is furthersupported by the fact that probe Sac 4.0 derived from SM-71 was able tohybridize to the corresponding region of KC-51 in Southern blotanalysis.

[0110] To determine the chromosomal origin of the Ig-unrelatedsequences, a recombinant phage (SM-71) derived from case SM1444, wasused as a probe in FISH analysis on metaphase chromosome spreads frommitogen-stimulated normal blood lymphocytes. The phage probe hybridizedspecifically to chromosome 14q32 as well as to chromosome 3q27 (FIG. 3),indicating that the recombinant phage insert contained one of the twochromosomal junctions of the reciprocal t(3;14) translocation. Thus,taken together, the results of cloning and FISH analysis establishedthat, in both NHL cases studied, the chromosomal translocation haslinked sequences within the switch region of the C_(μ) locus tosequences from band 3q27, consistent with the cytogenetic description ofthe t(3;14)(q27;q32) translocation. In the two NHL cases studied, thebreakpoints on 3q27 were located within 3 kb of the same genomic locus,which was termed bcl-6.

[0111] In order to determine whether 3q27 breakpoints in additional NHLcases were also located within the cloned portion of the bcl-6 locus,bcl-6 rearrangements were examined in a total of 19 NHL cases carrying3q27 breakpoints, including 4 (two cloned cases and two additional ones)carrying t(3;14)(q27;q32) as well as 15 cases carrying 3q27translocations involving regions other than 14q32. Southern blothybridization using probes derived from phage SM-71 (see FIG. 2)detected rearranged fragments in EcoRI- and/or BglII-digested DNA in 7of 19 cases studied, including all 4 t(3;14) cases as well as 3 caseswith other types of translocations (see FIGS. 4A-4C for cytogeneticdescription of the cases and representative results). These resultsindicate that heterogeneous 3q27 breakpoints cluster in a fairlyrestricted region within bcl-6 independently of the partner chromosomeinvolved in the translocation.

[0112] Whether the bcl-6 locus adjacent to the chromosomal breakpointscontained a transcriptional unit was investigated. Probe Sac 4.0 (seeFIG. 2) was used to detect RNA expression in several human cell lines byNorthern blot analysis. A major 2.4 kb RNA species was readilydetectable in two B-cell derived cell lines tested, while a relativelyless abundant 4.4 kb species is present in CB33 only. No hybridizationwas detected in a T-cell derived cell line (HUT 78) nor in HeLa cells(FIG. 5). This result indicates that 3q27 sequences immediately adjacentto the chromosomal breakpoint cluster are part of a gene (bcl-6) whichis expressed in cells of the B lineage.

Experimental Discussion

[0113] This study reports the identification and cloning of a genomicregion, bcl-6, involved in recurrent chromosomal translocationsaffecting band 3q27 in NHL. The region is defined by the clusteredposition of breakpoints in seven NHL cases carrying 3q27 translocationsinvolving either IgH or several other loci. A more precise definition ofthe bcl-6 locus and of the frequency of its involvement in NHL requirescloning and characterization of additional bcl-6 sequences and studyingadditional tumor cases. Nevertheless, the finding that varioustranslocation partner chromosomes have been joined to the same region onchromosome 3 in cytogenetically heterogenous NHL cases supports thenotion that rearrangement of the bcl-6 locus may represent the criticalcommon denominator of translocations involving 3q27.

[0114] The second finding of this study is that the bcl-6 locus containsa gene which is expressed in B-cells. It is not clear at this stagewhether the chromosomal breakpoints directly truncate coding orregulatory sequences of bcl-6, or, whether the gene remains intact withits regulation overridden by transcriptional control motifs juxtaposedby the translocation. The clustering of breakpoints in the seven studiedNHL cases suggests, however, that bcl-6 may be a proto-oncogene whichcan contribute to NHL pathogenesis upon activation by chromosomaltranslocation. Results of this study will allow elucidation of thenormal structure and function of the bcl-6 gene in order to understandthe pathogen consequences of chromosomal translocation of bcl-6 and itsrole in lymphomagenesis.

References for Section I

[0115] 1. Gaidano, G., and Dalla-Favera, R., (1992) Oncogenes and tumorsuppressor genes. In: Neoplastic Hematopathology, D. M. Knowles (ed.),Wilkins & Wilkins, pp 245-261.

[0116] 2. Dalla-Favera, R., et al. (1982) Human c-myc oncogene islocated on the region of chromosome 8 that is translocated in Burkittlymphoma cells, Proc. Natl. Acad. Sci. USA 79:7824-7827.

[0117] 3. Taub, R., et al. (1982) Translocation of c-myc gene into theimmunoglobulin heavy chain locus in human Burkitt lymphoma and murineplasmacytoma cells, Proc. Natl. Acad. Sci. USA 79:7837-7841.

[0118] 4. Bakhshi, A., et al. (1985) Cloning the chromosomal breakpointof t(14;18) human lymphomas: clustering around J_(H) on chromosome 14and near a transcriptional unit on 18, Cell 41:889-906.

[0119] 5. Tsujimoto, U., et al. (1985) Involvement of the Bcl-2 gene inhuman follicular lymphoma, Science 228:1440-1443.

[0120] 6. Cleary, M. L., and Sklar, J., (1985) Nucleotide sequence of at(14;18) chromosomal breakpoint in follicular lymphoma and demonstrationof a breakpoint-cluster region near a transcriptionally active locus onchromosome 18, Proc. Natl. Acad. Sci. USA 82:7439-7444.

[0121] 7. Motokura, T., et al. (1991) A novel cyclin encoded by a bcl-1linked candidate oncogene, Nature 350:512-514.

[0122] 8. Raffeld, M., and Jaffe, E. S., (1991) Bcl-l, t(11;14), andmantle zone lymphomas, Blood 78:259-261.

[0123] 9. Offit, K., et al. (1989) t(3;22)(q27;q11): A noveltranslocation associated with diffuse non-Hodgkin's lymphoma, Blood74:1876-1879.

[0124] 10. Bastard, C., et al. (1992) Translocations involving band 3q37and Ig gene regions in non-Hodgkin's lymphoma, Blood 79:2527-2531.

[0125] 11. Miller, S. A., et al. (1988) A simple salting out procedurefor extracting DNA from human nucleated cells, Nucleic Acids Res,16:1215-1218.

[0126] 12. Feinberg, A. P., and Vogelstein, B., (1983) A technique forradiolabelling DNA restriction endonuclease fragments to high specificactivity, Anal. Biochem., 132:6-13.

[0127] 13. Ravetch, J. V., et al. (1981) Structure of the humanimmunoglobulin μ locus: characterization of embryonic and rearranged Jand D regions, Cell, 27:583-591.

[0128] 14. Rao, P. H., et al. (1994) Subregional localization of 20single-copy loci to chromosome 6 by fluorescence in situ hybridization,Cyto. and Cell Genetics 66:272-273.

[0129] 15. Chirgwin, J. M., et al. (1979) Isolation of biologicallyactive ribonucleic acid from sources enriched in ribonuclease,Biochemistry, 18:5294-5299.

[0130] 16. Peschle, C., et al. (1984) Translocation and rearrangement ofc-myc into immunoglobulin alpha heavy chain locus in primary cells fromacute lymphocytic leukemia, Proc. Natl. Acad. Sci. U.S.A., 81:5514-5518.

[0131] 17. Showe, L. C., et al. (1985) Cloning and sequencing of a c-myconcogene in a Burkitt's lymphoma cell line that is translocated to agerm line alpha switch region, Mol. Cell. Biol., 5:501-509.

[0132] 18. Neri, A., Barriga, et al. (1988) Different regions of theimmunoglobulin heavy chain locus are involved in chromosomaltranslocations in distinct pathogenic forms of Burkitt lymphoma, Proc.Natl. Acad. Sci. USA, 85:2748-2752.

[0133] 19. Rabbits, T. H., et al. (1991) Human immunoglobulin heavychain genes: evolutionary comparisons of C mu, C delta and C gamma genesand associated switch sequences, Nucleic Acids Res., 9:4509-4524.

[0134] 20. Schmid, et al. (1991) Nature, 332:733.

Experimental Detail Section II Introduction

[0135] The molecular analysis of specific chromosomal translocations hasimproved the understanding of the pathogenesis of non-Hodgkin lymphoma(NHL), a heterogeneous group of B-cell or, less frequently, T-cellmalignancies (1,2). The (14;18) chromosomal translocation, which causesthe deregulated expression of the anti-apoptosis gene BCL-2, plays acritical role in the development of follicular lymphoma (FL) (3-6),which accounts for 20 to 30% of all NHL diagnoses (7). Burkitt'slymphoma (BL) and mantle-cell lymphoma, two relatively rare NHL types,are characterized by chromosomal translocations causing the deregulatedexpression of the cell-cycle progression genes C-MYC and theBCL-1/cyclin D1, respectively (8-15).

[0136] Relatively little is known about the molecular pathogenesis ofdiffuse large cell lymphoma (DLCL), the most frequent and most lethalhuman lymphoma (7). DLCL accounts for ˜40% of initial NHL diagnoses andis often the final stage of progression of FL(7) A small percentage ofDLCL display C-MYC rearrangements (16) and 20 to 30% display alterationsof BCL-2 reflecting the tumor's derivation from FL (17). However, noconsistent molecular alteration has been identified that is specific forDLCL.

[0137] Chromosomal translocations involving reciprocal recombinationsbetween band 3q27 and several other chromosomal sites are found in 8 to12% of NHL cases, particularly in DLCL (18-19). From NHL samplesdisplaying recombinations between 3q27 and the immunoglobulin (Ig) heavychain locus on 14q32, the chromosomal junctions of several(3;14)(q27;q32) translocations were cloned and identified a cluster ofbreakpoints at a 3q27 locus named BCL-6.

Experimental Results

[0138] To isolate normal BCL-6 cDNA, a cDNA library constructed form theNHL cell line Bjab (22) was screened with a probe (20-21) derived fromthe chromosomal region flanking the breakpoints of two t(3;14)(q27;32)cases. A phage cDNA library constructed from RNA of the Bjab lymphomacell line was screened (1×10⁶ plaques) by plaque hybridization with theSac 4.0 probe that had been ³²P-labelled by random priming (22).Sequence analysis (FIGS. 10A-10B) revealed that the longest clone (3549bp), approximately the same size as BCL-6 RNA, codes for a protein of706 amino acids with a predicted molecular mass of 79 kD. The putativeATG initiation codon at position 328 is surrounded by a Kozak consensussequence (23) and is preceded by three upstream in-frame stop codons.The 1101-bp 3′-untranslated region contains a polyadenylation signalfollowed by a track of poly(A). These features are consistent with BCL-6being a functional gene.

[0139] The NH₂- and COOH-termini of the BCL-6 protein (FIGS. 10A-10B)have homologies with “zinc-finger” transcription factors (24). BCL-6contains six C₂H₂ zinc-finger motifs (FIG. 10A) and a conserved stretchof six amino acids (the H/C link) connecting the successive zinc-fingerrepeats (25), BCL-6 can be assigned to the Krüppel-like subfamily ofzinc-finger proteins. The NH₂-terminal region of BCL-6 is devoid of theFAX (27) and KRAB (28) domains sometimes seen in Krüppel-relatedzinc-finger proteins, but it does have homologies (FIG. 11) with otherzinc-finger transcription factors including the human ZFPJS protein, aputative human transcription factor that regulates the majorhistocompatibility complex II promoter, the Tramtrack (ttk) andBroad-complex (Br-c) proteins in Drosophila that regulate developmentaltranscription (29), the human KUP protein (31), and the human PLZFprotein, which is occasionally involved in chromosomal translocations inhuman promyelocytic leukemia (32). The regions of NH₂-terminal homologyamong ZFPJS, ttk, Br-c, PLZF and BCL-6 also share some degree ofhomology with viral proteins (e.g. VASSR) of the poxvirus family (33) aswell as with the Drosophila kelch protein involved in nurse cell-oocyteinteraction (34). These structural homologies suggest that BCL-6 mayfunction as a DNA-binding transcription factor that regulates organdevelopment and tissue differentiation.

[0140] The cDNA clone was used as a probe to investigate BCL-6 RNAexpression in a variety of human cell lines by Northern blot analysis. Asingle 3.8 kb RNA species was readily detected (FIG. 11) in cell linesderived from mature B-cells, but not from pro-B-cells or plasma cells, Tcells or other hematopoietic cell lineages. The BCL-6 RNA was notdetectable in other normal other tissues, except for skeletal muscle inwhich low level expression was seen. Thus, the expression of BCL-6 wasdetected in B-cells at a differentiation stage corresponding to that ofDLCL cells. This selective expression in a “window” of B-celldifferentiation suggests that BCL-6 plays a role in the control ofnormal B-cell differentiation and lymphoid organ development.

[0141] To characterize the BCL-6 genomic locus, the same cDNA probe toscreen a genomic library from human placenta was used. A phage genomiclibrary constructed from normal human placenta DNA (Stratagene) wasscreened (8×10⁵ plaques) with the BCL-6 cDNA. Twelve overlapping clonesspanning 50 kb of genomic DNA were isolated. After restriction mapping,the position of various BCL-6 exons was determined by Southernhybridization using various cDNA probes. By restriction mapping,hybridization with various cDNA probes, and limited nucleotidesequencing, the BCL-6 gene was found to contain at least ten exonsspanning ˜26 kb of DNA (FIG. 12). Sequence analysis of the first andsecond exons indicated that they are noncoding and that the translationinitiation codon is within the third exon.

[0142] Various cDNA and genomic probes were used in Southern (DNA) blothybridizations to determine the relationship between 3q27 (Table 1).Monoallelic rearrangements of BCL-6 were detected in 12 of 17 tumors byusing combinations of restriction enzymes (Bam HI and Xba I) and probewhich explore ˜16 kb within the BCL-6 locus. These 12 positive casescarry recombinations between 3q27 and several different chromosomes(Table 1), indicating that heterogeneous 3q27 breakpoints cluster in arestricted genomic locus irrespective of the partner chromosome involvedin the translocation. Some DLCL samples (5 of 17) do not display BCL-6rearrangements despite cytogenetic alterations in band 3q27, suggestingthat another gene is involved or, more likely, that there are otherbreakpoint clusters 5′ or 3′ to BCL-6. If the latter is true, theobserved frequency of BCL-6 involvement in DLCL (33%, see below) may bean underestimate. TABLE 1 Frequency of BCL-6 rearrangements in DLCLcarrying chromosomal translocations affecting band 3q27 Fraction oftumors with BCL-6 Translocation rearrangements t(3;14(q27;q32) 4/4t(3;22)(q27;q11) 2/3 t(3;12)(q27;q11) 1/1 t(3;11)(q27;q13) 1/1t(3;9)(q27;p13) 0/1 t(3;12)(q27;q24) 0/1 der(3)t(3;5)(q27;q31) 1/1t(1;3)(q21;q27) 1/1 t(2;3)(q23;q27) 1/1 der(3)t(3;?)(q27;?) 1/3

[0143] A panel of tumors not previously selected on the basis of 3q27breakpoints but representative of the major subtypes of NHL as well asof other lymphoproliferative diseases was analyzed. Similarrearrangements were detected in 13 of 39 DLCL, but not in other casesincluding other NHL subtypes (28 FL, 20 BL, and 8 small lymphocyticNHL), acute lymphoblastic leukemia (ALL; 21 cases), and chroniclymphocytic leukemia (CLL; 31). These findings indicate that BCL-6rearrangements are specific for and frequent in DLCL. In addition, thefrequency of rearrangements in DLCL (33%) significantly exceeds that (8to 12%) reported at the cytogenetic level, suggesting that some of theobserved rearrangements may involve submicroscopic chromosomalalterations undetectable at the cytogenetic level.

[0144] All the breakpoints in BCL-6 mapped to the putative 5′ flankingregion, the first exon or the first intron (FIG. 12). For two patientsthat carry (3;12)(q27;q32) translocations, the chromosomal breakpointshave been cloned and precisely mapped to the first intron (SM1444) or to5′ flanking sequences (KC1445) of BCL-6 on 3q27, and to the switchregion of IgH on 14q32 (20-21). In all rearrangements, the coding regionof BCL-6 was left intact whereas the 5′ regulatory region, presumablycontaining the promoter sequences, was either completely removed ortruncated. The resultant fusion of BCL-6 coding sequences toheterologous (from other chromosomes) or alternative (within the BCl-6locus) regulatory sequences may disrupt the gene's normal expressionpattern. A BCL-6 transcript of normal size was detected by Northern blotanalysis of DLCL cells carrying either normal or truncated BCL-6. Someof the truncations were in the 5′ flanking sequences and would thereforenot be expected to generate structurally abnormal transcripts.

Experimental Discussion

[0145] Zinc-finger encoding genes are candidate oncogenes as they havebeen shown to participate in the control of cell proliferation,differentiation, and organ pattern formation (24). In fact, alterationsof zinc-finger genes have been detected in a variety of tumor types.These genes include PLZF (32) and PML (35-38) in acute promyelocticleukemia, EVI-1 (38-39) in mouse and human myeloid leukemia, TTG-1 (40)in T-cell CLL, HTRX (41-43) in acute mixed-lineage leukemia, and WT-1(44) in Wilm's tumor. Terminal differentiation of hematopoietic cells isassociated with the down-regulation of many Krüppel-type zinc-fingergenes. Thus, constitutive expression of BCL-6, caused by chromosomalrearrangements; interferes with normal B-cell differentiation, therebycontributing to the abnormal lymph node architecture typifying DLCL.

[0146] Given that DLCL accounts for ˜80% of NHL mortality (7), theidentification of a specific pathogenetic lesion has importantclinicopathologic implications. Lesions in BCL-6 may help in identifyingprognostically distinct subgroups of DLCL. In addition, since atherapeutic response can now be obtained in a substantial fraction ofcases (7), a genetic marker specific for the malignant clone may be acritical tool for the monitoring of minimal residual disease and earlydiagnosis of relapse (45).

[0147] The gene cloned from chromosomal translocations affecting band3q27, which are common in DLCL codes for a 79 kD protein that ishomologous with zinc-finger transcription factors. In 33% (13/39) ofDLCL samples, but no in other types of lymphoid malignancies, the BCL-6gene is truncated within its 5′ noncoding sequences, suggesting that itsexpression is deregulated. Thus, BCL-6 is a proto-oncogene specificallyinvolved in the pathogenesis of DLCL.

References for Section II

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[0170] 23. Kozak, M., (1989) J. Cell. Biol 108:229.

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[0174] 27. Knochel, W., et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6097.

[0175] 28. Bellefroid, E. J., et al. (1991) DNA 88:3608.

[0176] 29. Harrison, S.D., and Travers, A. A., (1990) EMBO J. 9:207.

[0177] 30. DiBello, R. R., et al. (1991) Genetics 129:385.

[0178] 31. Chardin, P., et al. (1991) Nucleaic Acid Res. 19:1431.

[0179] 32. Chen, Z., et al. (1993) EMBO J. 12:1161.

[0180] 33. Koonin, E. V., et al. (1992) Trends Biochem. Sci. 17:213.

[0181] 34. Xue, F., and Cooley, L., (1993) Cell 72:681.

[0182] 35. de Thé, H., et al. (1991) Cell 66:675.

[0183] 36. Kazizuka, A., et al. (1991) Cell 66:663.

[0184] 37. Pandolfi, P. P., et al. (1991) Oncogene 6:1285.

[0185] 38. Morishita, K., et al. (1988) Cell 54:831.

[0186] 39. Fichelson, S., et al. (1992) Leukemia 6:93.

[0187] 40. McGuire, E. A., et al. (1989) Cell Biol. 9:2124.

[0188] 41. Djabali, M., et al. (1992) Nature Genet. 2:113.

[0189] 42. Tkachuk, D. C., et al. (1992) Cell 71:691.

[0190] 43. Gu, Y., et al. (1992) Cell 71:701.

[0191] 44. Haber, D. A., et al. (1990) Cell 61:1257.

[0192] 45. Medeiros, L. J., et al. (1992) Neoplastic Hemopathology, pp.263-298.

Experimental Detail Section III Introduction

[0193] Non Hodgkin's lymphoma (NHL), the most frequent tumor occurringin patients between the ages of 20 and 40, includes several distinctclinico-pathologic subtypes, among which diffuse lymphoma with a largecell component (DLLC) is the most clinically relevant in terms ofmorbidity and mortality (1). DLLC include intermediate-grade lymphomaswith pure diffuse large-(DLCL), or mixed small- and large-cell (MX-D)histology, as well as high-grade immunoblastic (IMB) lymphoma. Thesetumors can occur “de novo”, accounting for 30-40% of initial NHLdiagnosis and, in addition, can represent the final “transformation”stage of follicular lymphomas (FL), small lymphocytic lymphoma andchronic lymphocytic leukemia. Considered together, “de novo” and“post-transformation” DLLC account for up to 80% of NHL mortality (1).

[0194] During the past decade, abnormalities involving proto-oncogenesand tumor suppressor genes have been identified in association withdistinct NHL subtypes (2). These genetic lesions represent importantsteps in lymphomagenesis as well as tumor-specific markers which havebeen exploited for diagnostic and prognostic purposes (3,4). Examplesinclude alterations of the MYC oncogene in Burkitt lymphoma (BL), and ofthe BCL-2 and BCL-1 oncogenes in FL and mantle-cell NHL, respectively.With respect to DLLC, several molecular alterations have been detectedat variable frequency, but none has been specifically or consistentlyassociated with the disease (2). In this invention the frequency anddisease-specificity of BCL-6 (5-10) rearrangements among the principalcategories of lymphoproliferative disease, including different NHLsubtypes, acute and chronic lymphoid leukemias and multiple myeloma isdemonstrated.

Materials and Methods

[0195] Samples of lymphnode biopsies, bone marrow aspirates andperipheral blood were collected by standard diagnostic procedures duringthe course of routine clinical evaluation in the Division of SurgicalPathology, Department of Pathology, Columbia University. In allinstances, the specimens were collected before specific anti-tumortreatment. Diagnoses were based on the results of histopathologic,immunophenotypic and immunogenotypic analysis (11). In all cases, thefraction of malignant cells in the pathologic specimen was at least 70%as determined by cytofluorimetric or immunohistochemical analysis ofcell-surface markers or antigen receptor (immunoglobulin heavy chain andT cell receptor β chain) gene rearrangement analysis (11).

[0196] Genomic DNA was prepared from diagnostic specimens by cell lysis,proteinase K digestion, phenol-chloroform extraction and ethanolprecipitation. For Southern blot analysis, 6 μg of DNA were digestedwith the appropriate restriction endonuclease, electrophoresed in a 0.8%agarose gel, denatured, neutralized and transferred to Duralose filters(Stratagene, La Jolla, Calif.). Filters were then hybridized with theBCL-6-specific Sac 4.0 probe (10) that had been ³²P-labelled by therandom priming technique. After hybridization, filters were washed in0.2× SSC (1× SSC=0.15M NaCl+ 0.015M sodium citrate/0.5% sodium dodecylsulfate) for 2 hours at 60° C. and then subjected to autoradiography for24-48 hours at −80° C. using intensifying screens.

[0197] All NHL cases were also analyzed for rearrangement of the BCL-2gene using the previously described probes corresponding to the MBR andMCR regions. Immunophenotypic analysis of immunoglobulin and cellsurface marker expression was performed as previously described (11).

[0198] Comparisons of histologic subsets with or without BCL-6rearrangement were made utilizing the method of inferences fromproportions (12).

Experimental Results

[0199] The tumor panel (Table 2) used for this study is representativeof the major categories of lymphoproliferative disease including NHL,125 cases, ALL 45, CLL 51 and MM 23. The NHL series was representativeof low-41, intermediate-45 and high-grade 24 subtypes according to theWorking Formulation. Fifteen cases of cutaneous T-cell NHL were alsoincluded.

[0200] The presence of BCL-6 rearrangements was analyzed by Southernblot hybridization of tumor DNAs using a probe (Sac 4.0) (10) andrestriction enzymes (BamHI and XbaI) which, in combination, explore aregion of 15.2 Kb containing the 5′ portion of the BCL-6 gene (firstexon, 7.5 Kb of first intron and 7.4 Kb of 5′ flanking sequences) (10).This region was previously shown to contain the cluster of breakpointsdetected in NHL. No additional rearrangements were found using probesand restriction enzymes exploring approximately 10 kb either 5′ or 3′ toBCL-6 sequences

[0201] The results of this analysis are summarized in Table 2 andrepresentatively shown in FIGS. 13A-13B. All cases of ALL, CLL and MMshowed a normal BCL-6 gene.

[0202] Eighteen of the 125 NHL cases displayed BCL-6 rearrangements.Among distinct NHL histologic subtypes, rearrangements were detected in16/45 (35.5%) DLLC and in 2/31 (6.4%) FL (p<0.001). One of these 2 FLcases showed both follicular and diffuse patterns of growth. Among DLLC,rearrangements were significantly more frequent in DLCL (15/33, 45.4%)than in MX-D (1/10, 10%) (p<0.01), suggesting that these genetic lesionsmay be specifically associated with the diffuse large cell component ofthese tumors. All of the DLLC cases displaying BCL-6 rearrangementslacked BCL-2 rearrangements which were found in only two 2 DLLC cases.Although cytogenetic data were not available for the panel of tumorsstudied, the frequency of BCL-6 rearrangements far exceeds that expectedfor 3q27 aberrations (10-12% in DLLC) (8, 9), suggesting that BCL-6rearrangements can occur as a consequence of submicroscopic chromosomalaberrations.

[0203] In order to determine whether the presence of BCL-6rearrangements correlated with distinct immunophenotypic features ofDLLC, the entire panel was analyzed for expression of immunoglobulin κand λ light chains, and B cell-associated antigens CD19, CD20 and CD22(11). As expected, the expression of these markers was variable in theDLLC cases tested. However, no correlation with the BCL-6 rearrangementstatus was found. TABLE 2 Rearrangements of the BCL-6 gene in lymphoidtumors TUMOR HISTOTYPE REARRANGED/TESTED % NHL Low grade: SL 0/10 0SCC-F 2*/18 11  MX-F 0/13 0 Intermediate grade: MX-D 1/10 10  DLCL 15/3345  SCC-D 0/2 0 High grade: IMB 0/2 0 SNCL 0/22 0 Others: CTCL 0/15 0ALL B-lineage: 0/34 0 T-lineage: 0/11 0 CLL B-lineage: 0/41 0 T-lineage:0/10 0 MM 0/23 0

Experimental Discussion

[0204] In this study, BCL-6 rearrangement is established as the mostfrequent abnormality detectable in DLLC. Previous studies have indicatedMYC and BCL-2 rearrangements detectable in 5-20% and 20% of DLLC,respectively (13). Compared to those lesions, which are also commonlyassociated with Burkitt's lymphoma (MYC) and FL (BCL-2), BCL-6rearrangements appear to be more disease-specific since they wereexclusively found in DLLC with the exception of 2 of 45 FL cases.Considering that one of these two FL cases displayed areas of diffusehistology, it is conceivable that BCL-6 rearrangements may beoccasionally associated with atypical FL cases with mixed follicular anddiffuse components. The recurrent and specific association between DLLCand structural lesions of a gene coding for a zinc finger-typetranscription factor related to several known proto-oncogenes 10suggests that these abnormalities may play a role in pathogenesis ofDLCL.

[0205] Among the heterogeneous DLLC spectrum, BCL-6 rearrangements weresignificantly more frequent in tumors displaying a pure diffuse largecell histology (DLCL) all of which lacked BCL-2 rearrangements.Considering that DLCL can originate both “de novo” and from the“transformation” of FL, and that the latter typically carry BCL-2rearrangements, results suggest that BCL-6 rearrangements may bespecifically involved in the pathogenesis of “de novo” DLLC. Thisconclusion is consistent with recent findings indicating that othergenetic alterations, namely the inactivation of the p53 tumor suppressorgene, may be involved in the transformation of FL to DLLC (14).

[0206] The results presented herein have relevant diagnostic andprognostic implications. DLLC represent a heterogeneous group ofneoplasms which are treated homogeneously despite the fact that only 50%of patients experience long-term disease free survival (1). The presenceof a marker such as BCL-6 rearrangement identifies a sizable subset ofcases with a distinct pathogenesis and, distinct biological behavior.

[0207] The pathogenesis of non-Hodgkin lymphoma with a large-cellcomponent (DLLC, including diffuse large-cell, DLCL, diffuse mixed-cell,MX-D, and immunoblastic, IMB) is unknown. The incidence anddisease-specificity of BCL-6 rearrangements in a large panel of lymphoidtumors, including acute and chronic lymphoid leukemias (96 cases),various NHL types (125 cases), and multiple myelomas (23 cases) has beentested. BCL-6 rearrangements were found in 16/45 (35.5%) DLLC, morefrequently in DLCL (15/33, 45%) than in MX-D (1/10, 10W), in 2/31 (6.4%)follicular NHL, and in no other tumor types. BCL-6 rearrangementsrepresent the first genetic lesion specifically and recurrentlyassociated with DLLC and should prove useful for understanding thepathogenesis as well as for the clinical monitoring of these tumors.

References for Section III

[0208] 1. Magrath, I. T. (1990) The Non-Hodgkin's Lymphomas: AnIntroduction, The Non-Hodgkin's Lymphomas, Edward Arnold, London, p 1.

[0209] 2. Gaidano, G., and Dalla-Favera, R. (1993) Biologic andmolecular characterization of non-Hodgkin's lymphoma, Curr. Opin. Oncol.5:776.

[0210] 3. Gribben, J. G., et al. (1991) Immunologic purging of marrowassessed by PCR before autologous bone marrow transplantation for B-celllymphoma, N. Engl. J. Med. 325:1525.

[0211] 4. Yunis, J. J., et al. (1989) Bcl-2 and other genomicalterations in the prognosis of large-cell lymphoma, N. Engl. J. Med.320:1047.

[0212] 5. Ye, B. H., et al. (1993) Cloning of bcl-6, the locus involvedin chromosome translocations affecting band 3q27 in B-cell lymphoma,Cancer Res. 53:2732.

[0213] 6. Baron, B. W., et al. (1993) Identification of the geneassociated with the recurring chromosomal translocationst(3;14)(q27;q32) and t(3;22)(q27;q11) in B-cell lymphomas, Proc. Natl.Acad. Sci. USA 90: 5262.

[0214] 7. Kerckaert, J. P., et al. (1993) LAZ3, a novel zinc-fingerencoding gene, is disrupted by recurring chromosome 3q27 translocationsin human lymphomas, Nature Genet. 5:66.

[0215] 8. Offit, K, et al. (1989) t(3;22 (q27;q11): A noveltranslocation associated with diffuse non-Hodgkin's lymphoma, Blood 74:1876.

[0216] 9. Bastard, C, et al. (1992) Translocations involving band 3q27and Ig gene regions in non-Hodgkin's lymphoma, Blood 79: 2527.

[0217] 10. Ye, B. H., et al. (1993) Alterations of a zinc-fingerencoding gene, BCL-6, in diffuse large-cell lymphoma, Science 262:747.

[0218] 11. Knowles, D. M. (ed.) (1992) Neoplastic Hemopathology,Williams & Wilkins, Baltimore, Md.

[0219] 12. Armitage, P., (1977) Statistical methods in medical research,Blackwell Scientific Publications, London, p. 11.

[0220] 13. Chaganti, R. S. K., et al. (1989) Specific translocations innon-Hodgkin's lymphoma: incidence, molecular detection, and histologicaland clinical correlations, Cancer Cells 7:33.

[0221] 14. Lo Coco, F., et al. (1993) p53 mutations are associated withhistologic transformation of follicular lymphoma, Blood 82:2289.

Experimental Detail Section IV Introduction

[0222] Non-Hodgkin lymphomas (NHL) represent one of the most commonmalignancies associated with human immunodeficiency virus (HIV)infection, and are recognized as an acquired immunodeficiency syndrome(AIDS)-defining condition (1-3). Since their initial observation in 1982(4), the incidence of AIDS-associated NHL (AIDS-NHL) has beenconsistently increasing (1, 2), and they now represent the most frequentHIV-associated malignancy in some AIDS risk groups, namely thehemophiliacs (5). Indeed, some estimates project that 10 to 20% of allnew NHL cases in the United States may eventually be related to AIDS(6).

[0223] AIDS-NHL are almost invariably B-cell derived NHL (1, 2, 7-12).When compared with NHL of similar histology arising in theimmunocompetent host, AIDS-NHL display distinctive clinical features,including late stage at presentation, poor prognosis, and the frequentinvolvement of extranodal sites (1, 2, 7-12). Systemic AIDS-NHL arehistologically heterogeneous, and have been initially classified intothree distinct categories, including small non cleaved cell lymphoma(SNCCL), large non cleaved cell lymphoma (LNCCL), and largecell-immunoblastic plasmacytoid lymphoma (LC-IBPL) (7, 9). Subsequently,most investigators have agreed to classify LNCCL and LC-IBPL as a singlecategory under the term of diffuse large cell lymphoma (DLCL).

[0224] Some progress has been made in elucidating the molecularpathogenesis of AIDS-SNCCL (1-3). AIDS-SNCCL is associated at variablefrequency with multiple genetic lesions, including Epstein Barr virus(EBV) infection, c-MYC translocation, RAS gene family mutation, and p53inactiviation by point mutation and allelic loss (1, 3, 13-25). On theother hand, the pathogenesis of AIDS-DLCL is relatively less defined.EBV infection appears to be the only genetic lesion associated with asignificant fraction of these tumors, particularly with the subsetdisplaying plasmacytoid features, p53 lesions have not been found andc-MYC activation is restricted to a small minority of cases (1-3,13-25).

Materials and Methods

[0225] Pathologic samples. Biopsy samples of lymph node, bone marrow,peripheral blood, or other involved organs from forty patients with AIDSwere collected during the course of standard diagnostic procedures.Thirty-two samples were derived from patients referred to the Departmentof Pathology, New York University, New York, N.Y. or to the Departmentof Pathology, Columbia University, New York, N.Y. Eight samples werederived from patients referred to the Departments of Hematology andPathology, University of Southern California School of Medicine, LosAngeles, Calif. Diagnosis was based on analysis of histopathology,immunophenotypic analysis of cell surface markers, and immunogenotypicanalysis of Immunoglobulin (Ig) gene rearrangement (32). In most cases,the fraction of malignant cells in the pathologic specimen was greaterthan 80%, as determined by cell suspension cytofluorometric or tissuesection immunohistochemical analysis of cell surface markers and by Iggene rearrangement analysis.

[0226] DNA extraction and Southern blot analysis. DNA was purified bydigestion with proteinase K, “salting out” extraction, and precipitationby ethanol (33). For Southern blot analysis (34), 6 μg of DNA wasdigested with the appropriate restriction endonuclease, electrophoresedin a 0.8% agarose gel, denatured, neutralized, transferred to Duralonfilters (Stratagene, LA Jolla, Calif.), and hybridized to probes whichhad been ³²P-labeled by the random primer extension method (35). Filterswere washed in 0.2× SSC (NaCl/Na citrate)/0.5% sodium dodecyl sulphate(SDS) for 2 hours at 60° C. and then autoradiographed using intensifyingscreens (Quanta III; Dupont, Boston, Mass.).

[0227] DNA probes. Immunoglobulin gene rearrangement analysis wasperformed using a J_(H) probe(36) (a gift of Dr. Korsmeyer) on HindIII,EcoRI, and BamHI digests. The organization of the BCL-6 locus wasinvestigated by hybridization of XbaI, BamHI, and BglII digested DNA tothe human BCL-6 probe Sac4.0 (26-27). In selected cases, a second proberepresentative of the BCL-6 locus, Sac0.8, was also used. Theorganization of the c-MYC locus was analyzed by hybridization of EcoRIand HindIII digested DNA to the human c-MYC locus was analyzed byhybridization of EcoRI and HindIII digested DNA to the human c-MYC probeMC413RC, representative of the third exon of the c-MYC gene (37). Thepresence of the EBV genome was investigated with a probe specific forthe EBV termini (5.2 Kb BamHI-EcoRI fragment isolated from the fusedBamHI terminal fragment NJ-het) (38).

Experimental Results

[0228] Forty cases of systemic AIDS-NHL were studied, including 13 SNCCLand 24 DLCL (8 LNCCL and 16 LC-IBPL). In addition, three cases of CD30+lymphomas, which have been sporadically reported in AIDS (39), were alsoincluded. All cases displayed a predominant monoclonal B-cell populationas determined by Ig gene rearrangement analysis.

[0229] Analysis of BCL-6 rearrangements. The BCL-6 gene contains atleast 9 exons spanning approximately 26 Kb of genomic DNA (27). Sequenceanalysis has shown that the first exon is non-coding and that thetranslation initiation codon is located within the third exon (27).Rearrangements of BCL-6 can be detected by Southern blot analysis usinga probe (Sac4.0) and restriction enzymes (BamHI and XbaI) which, incombination, explore a region of 15.2 Kb containing the 5′ portion ofthe BCL-6 gene (27) (FIGS. 14A-14C). This same region was previouslyshown to contain the cluster of chromosomal breakpoints detected in NHLof the immunocompetent host (27, 29). Cases showing an abnormallymigrating band in only one digest were further studied by hybridizingthe Sac4.0 probe to additional digests (BglII) or, alternatively, byhybridizing BamHI and XbaI digests to a probe (Sac0.8) derived from theBCL-6 first intron, which, being located 3′ of the breakpoint cluster,explores the reciprocal chromosome 3 (FIGS. 14A-14C). Only, casesshowing abnormally migrating bands with two restriction enzymes and/ortwo probes were scored as rearranged.

[0230] Rearrangements of BCL-6 were detected 5/24 AIDS-DLCL (20.8%),both in the LNCCL (2/8; 25%) and in the LC-IBPL (3/16; 18.7%) variants(Table 3 and FIGS. 14A-14C). All cases of AIDS-SNCCL and CD30+ lymphomasdisplayed a germline BCL-6 locus. The location of the breakpointsdetected in AIDS-HNL corresponds to the pattern most commonly observedin DLCL of the immunocompetent host. TABLE 3 Frequency of BCL-6rearrangements in AIDS-NHL DLCL^(b) SNCCL^(a) LNNCL LC-IBPL CD30 + NHLc0/13 2/8 3/16 0/3

[0231] Other genetic lesions. The other genetic lesions investigated inthe panel of AIDS-NHL included infection by EBV of the tumor clone,activation of the c-MYC and RAS proto-oncogenes, and inactivation of thep53 tumor suppressor gene. The experimental strategies used toinvestigate these lesions have been described in detail elsewhere (13,45, 40). For some of the cases, the molecular characterization of thesegenetic lesions have been previously reported (13, 14, 41); for theother cases, it has been assessed in the course of this study.

[0232] EBV infection was assessed by Southern blot hybridization using aprobe representative of the EBV termini (38) which allows to analyzeclonality in EBV-infected tissues (23) (FIGS. 16A-16C) . A monoclonalinfection was detected in 5/13 (38%) SNCCL, 17/24 DLCL (71%) [3/8(37.5%) LNCCL and 14/16 (87.5%) LC-IBPL], and 3/3 (100%) CD30+ cases.

[0233] Rearrangements of c-MYC were tested by hybridizing HindIII andEcoRI digested DNAs with a probe representative of c-MYC exon 3(41)(FIGS. 16A-16C). Rearrangements were present in 13/13 SNCCL (100%), 5/24(20.8%) DLCL [2/8 (25%) LNCCL and 3/16 (18.7%) LC-IBPL], and 2/3 CD30+cases.

[0234] Mutations of p53 and RAS were analyzed by a two step strategy.Single strand conformation polymorphism (SSCP) analysis was applied top53 exons 5 through 9 (in 29 cases) or p53 exons 5 through 8 (in 6cases) and to N-, K-, and H-RAS exons 1 and 2 (in 29 cases); casesdisplaying an altered electrophoretic pattern by SSCP were furtherstudied by DNA direct sequencing of the PCR product. p53 mutations werescored in 8/13 (61.5%) SNCCL, but in none of the DLCL tested (0/22).Finally, RAS activation by point mutation was positive in 3/13 (23%)SNCCL and in 1/16 (6%) DLCL tested.

[0235] The molecular features of the cases displaying BCL-6rearrangements are listed in Table 4. Overall, BCL-6 rearrangements weredetected both in the presence and in the absence of clonal EBV infectionof the tumor, whereas c-MYC alterations and p53 mutations wereconsistently absent in the cases displaying BCL-6 rearrangements. TABLEI Molecular features of AIDS-DLCL^(a) PATIENT HISTOL.^(b) CLONALITYBCL-6 EBV c-MYC p53 RAS DK782 LNCCL + + − − − − DK1178 LNCCL + + − − − −DK1028 LNCCL + − − − − − DK3973 LNCCL + − + − − − DK773 LNCCL + − − + −− RDF834 LNCCL + − + + − − DK1452 LNCCL + − − − − − DK64 LNCCL + − + −− + DK771 LC-IBPL + + + − − − K827 LC-IBPL + + + − − − DS16LC-IBPL + + + − − ND DK3537 LC-IBPL + − + + − − DK3357 LC-IBPL + − + − −− DK63 LC-IBPL + − + − − − DK1446 LC-IBPL + + + + − − DK3479 LC-IBPL + −− − − − DK2092 LC-IBPL + − + − − − DS17 LC-IBPL + − − − − ND DS45LC-IBPL ND − + − − ND DS46 LC-IBPL + − + + − ND DS93 LC-IBPL + − + − −ND DS136 LC-IBPL + − + − − ND DS155 LC-IBPL + − + − − ND DS165 LC-IBPL +− + − − ND

Experimental Discussion

[0236] Diffuse large cell lymphoma (DLCL) represents the most frequenttype of AIDS-NHL in the HIV-infected adult (8). Despite itsepidemiologic relevance, the molecular pathogenesis of these tumors islargely unclarified (3). Analysis of the genomic configuration of BCL-6in a panel of AIDS-NHL indicates that BCL-6 rearrangements are involvedin approximately 20% of AIDS-DLCL, whereas they are consistentlynegative in AIDS-SNCCL. In this respect, BCL-6 rearrangements may beconsidered the first identified genetic lesion specific for the DLCLtype among AIDS-NHL. BCL-6 rearrangements are present in both subgroupsof DLCL, i.e. LNCCL and LC-IBPL, and occur both in the absence and inthe presence of EBV infection of the tumor clone (Table 4). On the otherhand, BCL-6 rearrangements were never detected in AIDS-DLCL carryingc-MYC alterations (Table 4).

[0237] The molecular pathway leading to AIDS-SNCCL involves c-MYCrearrangements, p53 mutations, and EBV infection in 100%, 60%, and 40%of the cases, respectively (13-26). The presence of somatichypermutation in the immunoglobulin variable regions utilized byAIDS-SNCCL points to chronic antigen stimulation as an additionalmechanism in the development of these tumors. The second genetic pathwayis associated with AIDS-DLCL, involves EBV in the large majority ofcases, as well as c-MYC and/or BCL-6 rearrangements in a fraction ofcases (13-26). These distinct pathogenetic mechanisms correlate with anumber of clinical features which distinguish AIDS-SNCCL from AIDS-DLCL,including different age of onset and different CD4 counts at the time oflymphoma development (1,2,8).

[0238] Results suggest that the frequency of BCL-6 rearrangements inAIDS-DLCL is significantly lower than that in DLCL in theimmunocompetent host, where BCL-6 rearrangements occur in more than 40%of the cases. It is possible that the genetic pathogenesis of these twogroups of tumors is different, and that the molecular mechanisms activein AIDS-DLCL are characterized by a higher degree of heterogeneity.Among DLCL in the immunocompetent host, BCL-6 rearrangements areassociated with distinct clinical features, including the extranodalorigin of the lymphoma and the lack of bone marrow involvement. Inaddition, the presence of this rearrangement appears to represent afavorable prognostic marker.

References for Section IV

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[0246] 8. Beral, V., et al. (1991) AIDS-associated non-Hodgkin lymphoma,Lancet 337:805.

[0247] 9. Knowles, D. M., et al. (1988) Lymphoid neoplasia associatedwith the acquired immunodeficiency syndrome (AIDS), Ann. Int. Med.108:744.

[0248] 10. Levine, A. M., et al. (1984) Development of B-cell lymphomain homosexual men, Ann. Intern. Med. 100:7.

[0249] 11. Carbone, A., et al. (1991) A clinicopathologic study oflymphoid neoplasias associated with human immunodeficiency virusinfection in Italy, Cancer 68:842.

[0250] 12. Ioachim, H. L., et al. (1991) Acquired immunodeficiencysyndrome-associated lymphomas: Clinical, pathologic, immunologic, andviral characteristics of 111 cases, Hum. Pathol. 22:659.

[0251] 13. Ballerini, P., et al. (1993) Multiple genetic lesions inacquired immunodeficiency syndrome-related non-Hodgkin's lymphoma, Blood81:166.

[0252] 14. Gaidano, G., et al. (1993) In vitro establishment ofAIDS-related lymphoma cell lines: phenotypic characterization, oncogeneand tumor suppressor gene lesions, and heterogeneity in Epstein-Barrvirus infection, Leukemia 7:1621.

[0253] 15. Groopman, J. E., et al. (1986) Pathogenesis of B-celllymphoma in a patient with AIDS, Blood 67:612.

[0254] 16. Pelicci, P.-G., et al. (1986) Multiple monoclonal B cellexpansions and c-myc oncogene rearrangements in acquired immunedeficiency syndrome-related lymphoproliferative disorders. Implicationsfor lymphomagenesis, J. Exp. Med. 164:2049.

[0255] 17. Subar, M., et al. (1988) Frequent c-myc oncogene activationand infrequent presence of Epstein-Barr Virus genome in AIDS-associatedlymphoma, Blood 72:667.

[0256] 18. Haluska, F. G., et al. (1989) Molecular resemblance of anAIDS-associated lymphoma and endemic Burkitt lymphomas: implications fortheir pathogenesis, Proc. Natl. Acad. Sci. USA 86:8907.

[0257] 19. Meeker, T. C., et al. (1991) Evidence for molecular subtypesof HIV-associated lymphoma: division into peripheral monoclonal,polyclonal and central nervous system lymphoma, AIDS 5:669.

[0258] 20. Epstein-Barr virus and AIDS associated lymphomas. Editorial,Lancet 338:979, (1991).

[0259] 21. Hamilton-Dutoit, S. J., et al. (1991) Detection ofEpstein-Barr virus genomes in AIDS related lymphomas: sensitivity andspecificty of in situ hybridization compared with Southern blotting, J.Clin. Pathol. 44:676.

[0260] 22. Hamilton-Dutoit, S. J., et al. (1991) AIDS-related lymphoma.Histopathology, immunophenotype, and association with Epstein-Barr virusas demonstrated by in situ nucleic acid hybridization, Am. J. Pathol.138:149.

[0261] 23. Neri, A., et al. (1991) Epstein-Barr virus infection precedesclonal expansion in Burkitt's and acquired immunodeficiencysyndrome-associated lymphoma, Blood 77:1092.

[0262] 24. Nakamura, H., et al. (1993) Mutation and protein expressionof p53 in acquired immunodeficiency syndrome-related lymphomas, Blood82:920.

[0263] 25. Carbone, A., et al. (1993) Human immunodeficiencyvirus-associated systemic lymphomas may be subdivided into two maingroups according to Epstein-Barr viral latent gene expression, J. Clin.Oncol. 1:1674.

[0264] 26. Ye, B. H., et al. (1993) Cloning of BCL-6, the locus involvedin chromosome translocations affecting band 3q27 in B-cell lymphoma,Cancer Res. 53:2732.

[0265] 27. Ye, B. H., et al. (1993) Alterations of a zincfinger-encoding gene, BCL-6, in diffuse large cell-lymphoma, Science262:747.

[0266] 28. Baron, B. W., et al. (1993) Identification of the geneassociated with the recurring chromosomal translocationst(3;14)(q27;q32) and t(3;22) (q27;q11) in B-cell lymphomas, Proc. Natl.Acad. Sci. USA 90:5262.

[0267] 29. Kerckaert, J.-P., et al. (1993) LAZ3, a novel zinc-fingerencoding gene, is disrupted by recurring chromosome 3q27 translocationsin human lymphoma, Nature Genet. 5:66.

[0268] 30. Deweindt, C., et al. (1993) Cloning of a breakpoint clusterregion at band 3q27 involved in human non-Hodgkin's lymphoma, Genes.Chrom. & Cancer 8:149.

[0269] 31. Bastard, C., and Tilly, H. (1993) Response to letter “t(2;3)(p12;q27) in Hodgkin's disease of a human immunodeficiency-viruspositive patient with hemophilia”, by Schlegelberger B., Grote W.,Wacker H. H., Bartels H., Blood 81:265.

[0270] 32. Knowles, D. M., et al. (1986) T-cell receptor Beta chain (TB)gene rearrangements: genetic markers of T-cell lineage and clonality,Hum. Pathol. 17:546.

[0271] 33. Miller, S. A., et al. (1988) A simple salting out procedurefor extracting DNA from human nucleated cells, Nucleic Acid Res.16:1215.

[0272] 34. Sambrook, J., et al. (1989) Molecular cloning: a laboratorymanual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory.

[0273] 35. Feinberg, A. P., and Vogelstein, B. (1983) A technique forradiolabeling DNA restriction endonuclease fragments to high specificactivity, Anal. Biochem. 132:6.

[0274] 36. Korsmeyer, S. J., et al. (1981) Developmental hierarchy ofimmunoglobulin gene rearrangement in human leukemic pre-B cells, Proc.Natl. Acad. Sci. USA 78:7096.

[0275] 37. Dalla-Favera, R., et al. (1981) Human c-myc oncogene islocated on the region of chromosome 8 that is translocated in Burkittlymphoma cells, Proc. Natl. Acad. Sci. USA 78:7096.

[0276] 38. Weiss, L. M., et al. (1987) Epstein-Barr virus DNA in tissuesof Hodgkin's disease, Am. J. Path. 129:86.

[0277] 39. Chadburn, A., et al. (1993) CD30(Ki-1) positive anaplasticlarge cell lymphomas in individuals infected with the humanimmunodeficiency virus, Cancer 72:3078.

[0278] 40. Gaidano, G., et al. (1991) p53 mutations in human lymphoidmalignancies: Association with Burkitt lymphoma and chronic lymphocyticleukemia, Proc. Natl. Acad. Sci. USA 88:5413.

[0279] 41. Shibata, D., et al. (1993) Epstein-Barr virus-associatednon-Hodgkin's lymphoma in patients infected with the humanimmunodeficiency virus, Blood 81:2102.

Experimental Detail Section V Introduction

[0280] The group of diffuse lymphomas with a large cell component(DLLC), including diffuse mixed, immunoblastic, and large cell subtypes,and the group of follicular lymphomas, each comprise about 40 per centof non-Hodgkin's lymphomas (NEL) in this country (1). Together, theincidence of NHL is increasing at 3 to 4 per cont a year, a rate secondonly to that of malignant melanoma and lung cancer in women (2) Despitesignificant advances in treatment, approximately half of patients withDLLC will succomb to their disease, although “high risk” individuals maysuccessfully be treated by intensive chemotherapy and radiotherapyregimens including autologous bone marrow transplantation (3-7). Theformulation of prognostic models allow clinical trials to be directedtoward groups of patients with different risks for failure afterconventional treatment (5).

[0281] Cytogenetic studies as well as molecular genetic analysis ofalterations involving proto-oncogenes and tumor suppressor genes haveprovided insights into the pathogenesis of NHL, and have alsocontributed diagnostic and prognostic markers (8,9). Examples includerearrangements of the BCL-2 gene at 18q21 observed in up to 85 per centof follicular lymphomas, the BCL-1 gene at 11q13 rearranged inintermediate differentiation NHL, and the MYC gene, perturbed inBurkitt's lymphoma (8,9). While no recurring genetic abnormality hasbeen specifically associated with diffuse large cell lymphoma,rearrangement of BCL-2 has been observed in 20 to 30 per cent of cases,where it has been associated with decreased overall or disease freesurvival (10-12). Chromosomal translocations including those involvingthe MYC proto-oncogene, while noted in DLLC, were not as prognosticallysignificant as other recurring chromosomal abnormalities (8,13).

[0282] BCL-6 (14-19) rearrangement is found to denote a subset of DLLCcharacterized by extranodal presentation and a favorable clinicaloutcome. These results indicate that, in concert with other clinicalfeatures, this molecular marker may be utilized as a prognosticindicator at the time of diagnosis.

Materials and Methods

[0283] This study was comprised of 102 cases of DLLC studied atdiagnosis with documented clonal rearrangement of the IGH gene and DNAavailable for further analysis, derived form 229 DLLC seriallyascertained over a nine year period. Excluded were 127 cases studied atrelapse, T cell DLLC, or cases for which no DNA was available. For thisstudy, DLLC was defined as lymphomas of diffuse large cleaved,non-cleaved, immunoblastic, or mixed subtype, according to theInternational Working Formulation (20) as classified by ahematopathologist (DCS or DF). Cytogenetic analysis was attempted oneach of the specimens as previously described (21). For detection ofBCL-6 rearrangements, DNA from each case was digested with BamHI andXbaI and subjected to Southern blot analysis utilizing a 4 kb Sac1-Sac1fragment of the BCL-6 gene as a probe (19). Cases which did not yieldmetaphases for karyotypic analysis were also analyzed for rearrangementof the MBR and MCR breakpoint regions of the BCL-2 gene, as previouslydescribed (11). Aggregate descriptions of 47 of the cases in the currentseries were included in prior reports of cytogenetic abnormalities inDLLC (11, 13, 14). A detailed molecular analysis of 8 cases (nos. 352,755, 1098, 1254, 1403, 1444, 1445) demonstrating BCL-6 rearrangement hasbeen reported separately (19).

[0284] For each case, clinical data were compiled as previouslydescribed (22). Stage was assessed according to the modified Ann Arobrcriteria (25). For the purposes of separate evaluation of number ofextranodal sites of disease, radiographs or pathologic involvement ofthese sites were scored. In the quantitation of extranodal sites ofdisease as a prognostic variable, bone marrow, but not splenicinvolvement was scored, in accord with the International PrognosticIndex (5).

[0285] Clinical endpoints including complete response and freedom fromprogression were defined as previously described (3). Of 102 patientswith DLLC genetically analyzed prior to cytotoxic treatments, 93received systemic chemotherapy. Nine patients with early stage diseasewere treated by surgical resection and/or radiation therapy. Allpatients were treated with curative intent. Chemotherapy treatments wereclassified into three groups: NHL-4, CHOP and BACOP (1st generation);m-BACOD, NHL-7 (2nd generation); MCOP_B, NHL-9, NHL-14, NHL-15, L-20(3rd generation)(4,24,29). Eight patients expired before completion oftherapy, with incomplete staging evaluations, or of infectiouscomplications during or shortly after treatment. These cases wereconsidered not valuable for the determination of remission status, butwere included in the analysis of overall survival and freedom fromprogression. One patient was judged to be a complete remission which wasconfirmed by autopsy after expiration due to infectious complications 3weeks after completion of protocol treatment. All deaths, regardless ofcause were considered as endpoints in the analysis of overall survival.Median survival was determined by the method of Kaplan and Meler (30).Analysis of correlations between gene rearrangements and clinicalfeatures were performed utilizing Fisher's exact test (13). Means werecompared utilizing two sample t-tests. Univariate comparisons ofsurvival and duration free from progression were made by log rank test.Survival and freedom from progression estimates are quoted withconfidence intervals (CI) given in parentheses. Multivariate analysiswas performed utilizing the Cox regression model (31). Stepwise multiplelogistic regression was sued in the multivariate analysis of factorsprognostic for achieving a complete response. For all statisticalanalyses, a P<0.05 based on a 2-sided test was considered significant.

Experimental Results

[0286] Of 102 cases of DLLC studied at diagnosis, 23 demonstrated BCL-6rearrangement, 21 demonstrated t(14;18) or rearrangement of BCL-2, and58 demonstrated no evidence of either BCL-6 or BCL-2 rearrangement.Representative results of hybridization analysis for rearrangement ofBCL-6 are depicted in FIGS. 17A-17B. The clinical characteristics ofgroups according to BCL-6 or BCL-2 rearrangement are summarized in Table5. The histologic subtypes and clinical features of the BCL-6 rearrangedcases are shown in Table 6.

[0287] The key to Table 6 is as follows: +E,uns Underlining signifiessite from which biopsy was performed. Histology: DLC=diffuse large cell;IMB immunoblastic; DML=diffuse mixed lymphoma; LDH= lactatedehydrogenase in units/ml; (B)=bulky disease (>8 cm or ⅓ thoracicdiameter); CHOP= cyclophosphamide, daunorubicin, vincristine,prednisone; MACOPB=methotrexate, daunorubicin, cyclophosphamide,vincristine, prednisone, bleomycin; MBACOD=same drugs as MACOPB withdexamethasone instead of prednixone and drugs in different schedule;PrCyBom—drugs of MACOPB plus cytosine arabinoside, etopiside,methotrexate; L-20-vincristine, cyclophosphamide, methotrexate,daunorubicin, prednisone, cytosine arabinoside, 1-asparaginase, BCNU,6-mercaptopurine, dactinomycin; 1-20 includes randomization toautologous transplantation;. NHL-7= CHOP plus methyl GAG, etoposide(36),NHL-14 short course PrCyBom; NHL-15—high dose daunorubicin, vincristine,cyclophosphamide(29); RT=radiation therapy; SURG=surgery CR=completeresponse; sCR= surgical complete response (all evaluable diseaseresected); PR=partial response; “+”=alive at last follow-up; e=expired;rel=relapse; NE=not evaluable; =Patient 1445 had a history of low gradeNHL of eyelid 7 years earlier, treated by radiotherapy. Skin involvementof patient 252 was not noted in a prior report (14) and patient 1445 hada history of low grade NHL of eyelid 7 years earlier, treated byradiotherapy.

[0288] While each of the BCL-6 rearranged cases was classified as aDLLC, the range of morphologies included diffuse large cell (cleaved andnon-cleaved), and less frequently, immunoblastic, or mixed histologies.Extensive necrosis and extranodal extension were common histologicfeatures, and were present in one of two cases of BCL-6 rearrangementwhich did not show clinical evidence of extranodal disease.

[0289] The BCL-6 rearranged cases had a mean age of 64.1 years atpresentation and a high frequency of extranodal involvement by disease;19 of 23 cases had stages IE, IIE, IIIE or stage IV desease, compared to48 of 79 of BCL-6 germline cases (p+0.07). Extranodal sites includedmuscle or subcutaneous tissues (6 cases), stomach (5 cases), lung orpleura (5 cases), skin, breast, bowel, thyroid, pancreas, or kidney, asassessed by biopsy or radiographic abnormalities which improved afterchemotherapy. Of the 7 cases with state IE or IIE decrease, 5 wereprimary extranodal lymphomas, while 2 were extranodal extensions from aprimary nodal site. Two cases were primary splenic lymphomas. In twocases, there was only peripheral adenopathy. Compared to BCL-6 germlinecases, there was no significant difference in the proportion of BCL-6rearranged cases with stage IV disease. Bone marrow involvement wasobserved in 15 of 75 BCL-6 germline cases biopsied, compared to only 1of the 23 stage IV BCL-6 rearranged cases (P=0.1).

[0290] All but one of the 23 patients with BCL-6 rearrangement at thetime of diagnosis received anthracycline-containing chemotherapy. Thispatient remained free of disease eight years after resection of aprimary splenic large cell lymphoma. At median follow-up in excess oftwo years, 21 of the 23 patients with BCL-6 rearrangement survived; theactuarial survival was 91 per cent (CI 80 per cent to 100 per cent). Twopatients expired during or immediately following treatment; an autopsyin one case revealed no evidence of lymphoma. This patient, and 19others were judged to have achieved a complete remission aftertreatment. Two patients relapsed with recurrent disease in the lung andtwo patients had persistent subcutaneous masses. One of the relapsepatients (case 295) went on to autologous transplanation and remainsfree of disease 78 months post-transplant.

[0291] With respect to known prognostic variables, the proportion of theBCL-6 rearranged cohort with LDH > 500 U per liter was similar to theproportion of the BCL-6 germline DLLC (3/23 versus 13/79; P=0.99). Fiveof 23 cases of DLLC with BCL-6 rearrangement demonstrated bulky disease,compared to 35 of 79 cases without BCL-6 rearrangement (P=0.1). Theproportion of cases with “limited stage” (I, IE, II, or IIE) disease wascomparable in the cohorts with and without BCL-6 rearrangement (Table5). TABLE 5 Characteristics of 102 cases of DLLC BCL-6- BCL-6+ BCL-6+BCL2- BCL2+ n = 23 58 21 Mean age (years) 64.1 52.7 62.8 Mean lactate405 331 389 dehydrogenase (U/ml) Mean extranodal sites 1.6 .93 .81 Bonemarrow 1/23 8/54 7/21 involvement Stage I(IE) 3(1) 3(3) 0 II(IIE) 7(6)22(6) 5 III(IIIE) 2(1) 8(2) 4 IV 11 25 12 Histology Diffuse large cell20 53 19 Diffuse mixed 1 2 1 Immunoblastic 2 3 1 Treatment 1stgeneration chemo. 12 16 10 2nd generation chemo. 1 10 3 3rd generationchemo. 9 24 8 other 1 8 0 Complete Remission 20/23 35/50 15/21 Rate 86%70% 71% Projected survival 91% 59% 46% at 36 months (CI (CI (CI80%-100%) 44%-74%) 21%-72%) Projected freedom 82% 56% 31% fromprogression (CI (CI (CI at 36 months 66%-98%) 43%-70%) 8%-53%)

[0292] TABLE 6 Clinical features of 23 cases of DLLC with BCL6rearrangement CASE AGE/ EXTRANODAL LDH CLINICAL NUMBER SEX STAGE SITESHISTOLOGY (BULK) TREATMENT OUTCOME 102 66/F IIS spleen DLC 3624 SURG,CHOP CR.96+ 147 61/F IS spleen DLC 365 SURG, RT sCR.101+ (B) 252 54/M IVspleen, skin DLC 126 MACOPB CR.88+ 278 68/M IV pleura, DLC 235 MACOPBPR.6e iliac mass (B) 295 46/M IV lung DLC 179 MACOPB, L-20 CR. rel, 97+352 53/F IV stomach, liver, IMB 775 MACOPB CR.81+ spleen, small (B)bowel, pleural effasion 470 74/F IV lung DLC 224 CHOP CR,30+ 534 70/FIIES spleen, mass DLC 278 CHOP CR,80+ involving pancreas (B) 763 79/FIIE stomach DLC 196 CHOP CR,60+ 970 75/M IV kidney, DLC 240 NNL-14 CR,4estomach 1020 60/M IIIE tonsil, DLC 303 CHOP CR,100+ pancreas 1056 63/MIIE stomach DLC 213 SURG, MBACOD CR,100+ 1058 59/M IIE axillary mass DLC206 PrCyBom CR,37+ involving breast 1098 74/F IV subcutaneous DLC 181RT, CHOP PR,36+ masses 1189 71/M IV subcutaneous DLC 330 CHOP PR,21+masses 1254 74/F IIE tyroid DLC 196 CHOP/RT CR,27+ 1264 76/F IV Lung,liver, DLC 236 CHOP CR,rel,27+ spleen, kidney 1299 50/M IE deltoid mass,DLC 529 PrCyBom CR,16+ bone (B) 1363 47/M III NONE DLC 129 NHL-15 CR,16+1403 62/M I NONE IMB 222 CHOP, RT CR,11+ 1407 71/M IIE stomach DLC 206SURG, CHOP CR,12+ 1444 70/F IV lung DLC 150 CHOP CR,14+ 1445 63/F IVneck mass DLC 174 NHL-15 CR,8+ involving muscle*, bone marrow

[0293] Multivariate analysts of clinical outcome. The median durationfree from progression of disease was not reached in the BCL-6 rearrangedDLLC compared to 70 months for BCL-6 germline cases, regardless of BCL-2status (P=0.009) (FIG. 18A). Projected freedom from progression at 36months was 82% (CI 66%-98%) and 49% (CI 37%-60%), respectively.Multivariate analysis revealed that four variables, BCL-6 status, stageIV disease, bulk of disease, and LDH (log transformed) were the mostpowerful prognostic indicators for freedom from progression (Table 7).Multivariate analysis of survival demonstrated that bulk, LDH, BCL-6status, and stage IV disease were the most useful predictors of overallsurvival (P=0.01, P=0.02, P=0.02, P=0.05, respectively). TABLE 7Multivariate analysis of freedom from progression Variables selectedinto Cox regression Relative P value model Risk (Wald chi square) BCL-6rearranged 0.18 (CI).04-.78) 0.007 Bulky disease 2.4 (CI 1.3-7.4) 0.01Stage IV disease 2.1 (CI 0.98-5.2) 0.03 LDH (log transformed) 1.6 (CI1.1-3.9) 0.05

[0294] The prognostic value of BCL-6 gene status was compared to riskvariables calculated according to the International Prognostic Index⁵,including serum LDH level, stage, performance status, and number ofextranodal sites. A cox regression analysis confirmed the independentprognostic value of BCL-6 gene status; patients with BCL-6 rearrangementhad a relative risk (RR) of dying of 0.09 (CI 0.02 to 0.42) compared topatients without BCL-6 rearrangement, controlling for the otherprognostic variables in the model (P=0.002).

[0295] When cases were considered with respect to BCL-2 status, theBCL-2 rearranged cases demonstrated a trend for a decreased survivalcompared to BCL-2 germline cases, regardless of BCL-6 status (P=0.12).When BCL-6 and BCL-2 status were considered together (FIG. 18B), BCL-6rearranged cases demonstrated a projected actuarial survival at 36months of 91% (CI 80%-100%) compared to 59% (CI 44%-74%) for the BCL-6germline/BCL-2 germline cohort, and 46% (CI 21%-72%) for the BCL-2rearranged cohort. While the logrank test between these three cohortsdemonstrated a difference in survival (P=0.02, FIG. 18B), the majorfactor driving the significant summary P value was the better survivalof the BCL-6 rearranged cohort. The projected freedom from progressionat 36 months was 82% (CI 66%-98%), 56% (CI 43%-70%) and 31% (CI 8%-53%)for the three groups. Median follow-up for survivors was two years.BCL-2 rearrangement did not emerge as an independent prognostic markerin the multivariate analysis of survival or freedom from progression.

[0296] There was also no prognostically significant effect of generationof chemotherapy treatment on survival, or freedom from progression(P=0.95, 0.21, respectively). There was a trend for a higher completeresponse rate among the BCL-6 rearranged cohort (Table 5, P=0.1),although logistic regression revealed that only the clinical parametersLDH, stage IV, and bulk of disease were independent predictors ofresponse.

[0297] Relationship between BCL-6, BCL-2, and 8q24 rearrangements. Ofthe 79 cases which lacked BCL-6 rearrangement, 21 demonstratedt(14;18)(q32;q21) or rearrangement of BCL-2 by molecular analysis. Thesecases were characterized by an older age at diagnosis, but were similarto the larger cohort of BCL-2 negative, BCL-6 negative cases withrespect to LDH, and distribution of histologies (Table 6).

[0298] Nine cases of DLLC demonstrated t(8;14)(q24;q32). Three of thesebiopsies were from extranodal sites including liver, bone and softtissue. Two additional cases were splenic lymphomas. In two cases,t(8;14) bearing DLLC also demonstrated BCL-6 rearrangement. There was noimpact on survival of the t(8;14) in DLLC with or without BCL-6rearrangement. The two cases of t(8;14) with co-incident BCL-6rearrangement did not show evidence of histologic transformation orother unusual histologic features. One of these cases (no. 147) was thesingle case treated by splenectomy and radiation therapy alone. Thesecond case was the single BCL-6 rearranged case successfully salvagedby autologous transplantation.

[0299] Cytogenetic features, including the relationship between 3q27 andBCL-6 rearrangement. Of the 65 DLLC with karyotypic abnormalities, 14demonstrated translocations and one a deletion affecting band 3q27; only11 among these 15 cases showed rearrangement of BCL-6 Five cases withapparently normal chromosomes 3 demonstrated BCL-6 rearrangements by DNAanalysis.

Experimental Discussion

[0300] As a group, DLLC are among the most common forms of NHL seen inthis country (1). These tumors have not, however, been associated with acharacteristic genetic abnormality (8). Seen in the vast majority offollicular lymphomas, t(14;18)(q32;q21) or its molecular equivalent,BCL-2 rearrangement, have been observed in 20 to 30 per cent of DLLC(8). In such cases, the t(14;18) may reflect a follicular origin ofthese tumors. The recognition of translocations involving 3q27 and thesites of IG genes, 14q32, 22q11, and 2p12, in predominantly diffuse NHLled to the molecular cloning of BCL-6 (14-19). While not unique todiffuse large cell lymphomas, translocations affecting 3q27 wereobserved only in 7 of >200 cases off follicular NHL with abnormalkaryotype reported in catalog of chromosome abnormalities in cancer(32). Of 28 cases of follicular NHL analyzed in a prior study, nonedemonstrated rearrangement of BCL-6 (19). BCL-6 rearrangement wasestablished as the most common genetic lesion specific to DLLC at thetime of diagnosis.

[0301] Unlike 18q21 translocations in NHL which, to date only haveinvolved IG gene loci as reciprocal partners, 3q27 translocationsdemonstrated a marked promiscuity of rearrangement partners. In additionto the sites of the IG genes, reciprocal translocations involving the3q27-29 region with at least 12 other loci; a total of 79 DLLC with 3q27translocations has been demonstrated.

[0302] Since 4 tumors in the current series with documented 3q27aberrations did not reveal BCL-6 rearrangement with the probe used inthis study, the true frequency of BCL-6 rearrangement in DLLC atdiagnosis may be higher than the 23 per cent rate reported here.Additional breakpoints may be documented outside the recognized breakcluster region of BCL-6 (19), in neighboring genes such as EV-1 (34), orin other genes not yet described. Such molecular heterogeneity is notunique in NH1; seemingly identical chromosomal translocations have beenshown to demonstrate a diversity of breakpoints possibly involvingdifferent genes (35).

[0303] The frequent occurrence of BCL-6 rearrangement in DLLCcharacterized by extranodal involvement represents one of the fewgenetic markers for this subset of lymphoma (8). Rearrangements ofBCL-1, BCL-2, or BCL-3 have been documented infrequently in extranodallymphomas (36-38), while 5 of 12 gastric lymphomas in one seriesdemonstrated MYC (8q24) rearrangement (38). The current series did notconfirm the association between 8q24 rearrangement and gastric lymphoma,although t(8;14) was seen in five cases of extranodal lymphoma, one ofwhich also showed BCL-6 rearrangement. The proportion of BCL-6rearranged cases with stages IE, IIE, IIIE, or IV disease was higherthan the proportion of BCL-6 germline DLLC; in the latter group, stageIV disease was more commonly due to bone marrow involvement. Whetherthis association with extranodal involvement of disease reflects aneffect of the primary deregulation of BCL-6 or “secondary” geneticevents associated with tumor progression (8,21) is unclear. Theobservation of t(3;22), t(2;3), or t(3;14) as solitary cytogeneticabnormalities in some tumors (14,15), is consistent with a primarypathogenetic role for this translocation.

[0304] While this analysis and two other reports did not confirm thevery short survival of BCL-2 rearranged DLLC initially reported (10, 12,13, 39), the BCL-2 rearranged DLLC did demonstrate a trend for a pooreroverall survival. The finding of a favorable prognosis for the subset ofstage IE-IIE extranodal DLLC with BCL-6 rearrangement is consistent withprior reports of a good prognosis associated with localized extranodallarge cell NHL treated with surgery or radiotherapy (40). Extranodalinvolvement in advanced stage disease, noted in the majority of theBCL-6 rearranged cases, has generally been considered a poor prognosticfactor in large series of DLLC, although the negative impact of thisfeature was most evident when combined with other adverse indicatorssuch as bulk, high LDH, or low performance status (5, 22, 41). Incontrast, bone marrow involvement, observed in 22 percent of DLLC, andconsidered an extranodal site in the International Prognostic Index (5),was rare in the BCL-6 rearranged cohort. The favorable treatment outcomeof the BCL-6 cohort, must also be tempered by the observation of relapseor residual disease in 3 of the patients still alive. An additionalrelapse case remains in remission 6 years after “salvage” autologoustransplantation.

[0305] The BCL-6 rearranged cohort of DLLC also possessed other clinicalmarkers of favorable prognosis; although comparable with respect to LDHand proportion with stage I-IIIE disease, the proportion of cases withbulky disease or bone marrow involvement was lower in the BCL-6rearranged cohort. Multivariate analysis suggested, however, that BCL-6gene rearrangement added independent prognostic power when analyzedtogether with clinically-derived variables of the InternationalPrognostic Index (5). This observation is illustrated by case 352, whichdisplayed both BCL-6 rearrangement as well as clinical featuresconsistent with a high level of risk in the International Index(elevated LDH, extensive extranodal involvement, low performance status,and stage IV disease), but who attained a durable remission.

[0306] Because of issues of toxicity versus efficacy of autologous bonemarrow transplantation or peripheral stem cell rescue, theidentification of both favorable and unfavorable prognostic markersoffers the potential to stratify treatment approaches to DLLC based onrisk groups (4-7, 22, 41). The probability of treatment failure remainsas high as 25-40 per cent for the most favorable subsets of DLLC basedon current prognostic models, highlighting the need for genetic or otherprognostic markers (5). In addition to its potential diagnostic andprognostic applications, the further identification of BCL-6 breakpointregions offers the opportunity to develop new polymerase chainreaction-derived measures of minimal residual disease (43). Theavailability of BCL-6 rearrangement as a new molecular marker of largecell lymphoma constitutes a potentially important clinical tool in themanagement of patients with this desease.

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[0333] 26. Warrell, R. P., et al. (1989) Short term intensive treatmentof intermediate-grade non-Hodkin's lymphoma using infusionalchemotherapy, Proc. Annu. Meet. Am. Soc. Clin. Oncol. 8:1054.

[0334] 27. Straus, D. H., et al. (1991) Small non-Cleaved cell lymphoma(undifferentiated lymphoma/Burkitt's type) in American adults. Resultswith treatment designed for acute lymphoblastic leukemia in adults, Am.J. Med. 90:328-337.

[0335] 28. O'Brien, J., et al. (1992) NHL-15 protocol for diffuseaggressive lymphomas: a dose intense regimen of doxorubicin,vincristine, and cyclophosphamide, Blood 80:157a.

[0336] 29. Kaplan, E. L., and Meier, P. (1958) Nonparametric estimationfrom incomplete observations, J. Am. Stat. Soc. 53:457-81.

[0337] 30. Cox, D. R. Regression models and life tables, J. R. Stat.Soc. B. 34:187-220.

[0338] 31. Mitelman, F. (1991) Catalog of chromosome aberrations incancer, New York.

[0339] 32. Leroux, D., et al. (1990) Translocation t(3;22)(q27;q11) inthree patients with diffuse large B cell lymphoma, Leukemia 4:373-376.

[0340] 33. Fichelson, S., et al. (1992) Evi-1 expression in leukemicpatients with rearrangements of the 3q25-q28 chromosomal region,Leukemia 6:93-99.

[0341] 34. Ladanyi M, et al. (1992) Follicular lymphoma witht(8;14)(q24;q32). A distinct clinical and molecular subset oft(8;14)-bearing lymphomas, Blood 79:2124-30.

[0342] 35. Raghoebier, S., et al. (1991) Essential differences inoncogene involvement between primary nodal and extranodal large celllymphoma, Blood 78:2680-86.

[0343] 36. Clark, H. M., et al. (1992) Cytogenetic and molecular studiesof t(8;14) and t(14;19) in nodal and extranodal b:cell lymphoma, J.Pathol. 166:129-37.

[0344] 37. Van Krieken, J. H. J. M., et al. (1991) Molecular genetics ofgastrointestinal non-Hodkin's lymphomas: unusual prevalence and patternof c-myc rearrangements in aggressive lymphomas, Blood 76:797-800.

[0345] 38. Romaguera, J. E., et al. (1993) The clinical relevance oft(14;18)/bcl-2 rearrangement and del 6q in diffuse large cell lymphomaand immunoblastic lymphoma, Ann. Oncol. 4:51-4.

[0346] 39. Rudders, R. A., et al. (1978) Primary extranodal lymphoma:Response to treatment and factors influencing prognosis. Cancer42:406-16.

[0347] 40. Coiffier, B., et al. (1991) Prognostic factors in aggressivemalignant lymphomas: description and validation of a prognostic indexthat could identify patients requiring a more intensive therapy, J.Clin. Oncol. 9:211-19.

[0348] 41. Schneider, A. M., et al. (1990) Treatment results with anaggressive chemotherapeutic regimen (MACOP-B) for intermediate and somehigh grade non-Hodkin's lymphomas, J. Clin. Oncol. 8:94-102.

[0349] 42. Gribben, J. G., et al. (1993) Detection by polymerase chainreaction of residual cells with the BCl-2 translocation is associatedwith increased risk of relapse after autologous bone marrowtransplantation for b-cell lymphoma, Blood 81:3449-87.

1 9 3720 base pairs nucleic acid double linear cDNA not provided CDS328..2445 1 GGCCCCTCGA GCCTCGAACC GGAACCTCCA AATCCGAGAC GCTCTGCTTATGAGGACCTC 60 GAAATATGCC GGCCAGTGAA AAAATCTTAT GGCTTTGAGG GCTTTTGGTTGGCCAGGGG 120 AGTAAAAATC TCGGAGAGCT GACACCAAGT CCTCCCCTGC CACGTAGCAGTGGTAAAGT 180 CGAAGCTCAA ATTCCGAGAA TTGAGCTCTG TTGATTCTTA GAACTGGGGTTCTTAGAAG 240 GGTGATGCAA GAAGTTTCTA GGAAAGGCCG GACACCAGGT TTTGAGCAAAATTTTGGAC 300 GTGAAGCAAG GCATTGGTGA AGACAAA ATG GCC TCG CCG GCT GAC AGCTGT 351 Met Ala Ser Pro Ala Asp Ser Cys 1 5 ATC CAG TTC ACC CGC CAT GCCAGG GAT GTT CTT CTC AAC CTT AAT CGT 399 Ile Gln Phe Thr Arg His Ala ArgAsp Val Leu Leu Asn Leu Asn Arg 10 15 20 CTC CGG AGT CGA GAC ATC TTG ACTGAT GTT GTC ATT GTT GTG AGC CGT 447 Leu Arg Ser Arg Asp Ile Leu Thr AspVal Val Ile Val Val Ser Arg 25 30 35 40 GAG CAG TTT AGA GCC CAT AAA ACGGTC CTC ATG GCC TGG AGA GGC CTG 495 Glu Gln Phe Arg Ala His Lys Thr ValLeu Met Ala Trp Arg Gly Leu 45 50 55 TTC TAT AGC ATC TTT ACA GAC CAG TTGAAA TGC AAC CTT AGT GTG ATC 543 Phe Tyr Ser Ile Phe Thr Asp Gln Leu LysCys Asn Leu Ser Val Ile 60 65 70 AAT CTA GAT CCT GAG ATC AAC CCT GAG GGATTC TGC ATC CTC CTG GAC 591 Asn Leu Asp Pro Glu Ile Asn Pro Glu Gly PheCys Ile Leu Leu Asp 75 80 85 TTC ATG TAC ACA TCT CGG CTC AAT TTG CGG GAGGGC AAC ATC ATG GCT 639 Phe Met Tyr Thr Ser Arg Leu Asn Leu Arg Glu GlyAsn Ile Met Ala 90 95 100 GTG ATG GCC ACG GCT ATG TAC CTG CAG ATG GAGCAT GTT GTG GAC ACT 687 Val Met Ala Thr Ala Met Tyr Leu Gln Met Glu HisVal Val Asp Thr 105 110 115 120 TGC CGG AAG TTT ATT AAG GCC AGT GAA GCAGAG ATG GTT TCT GCC ATC 735 Cys Arg Lys Phe Ile Lys Ala Ser Glu Ala GluMet Val Ser Ala Ile 125 130 135 AAG CCT CCT CGT GAA GAG TTC CTC AAC AGCCGG ATG CTG ATG CCC CAA 783 Lys Pro Pro Arg Glu Glu Phe Leu Asn Ser ArgMet Leu Met Pro Gln 140 145 150 GAC ATC ATG GCC TAT CGG GGT CGT GAG GTGGTG GAG AAC AAC CTG CCA 831 Asp Ile Met Ala Tyr Arg Gly Arg Glu Val ValGlu Asn Asn Leu Pro 155 160 165 CTG AGG AGC GCC CCT GGG TGT GAG AGC AGAGCC TTT GCC CCC AGC CTG 879 Leu Arg Ser Ala Pro Gly Cys Glu Ser Arg AlaPhe Ala Pro Ser Leu 170 175 180 TAC AGT GGC CTG TCC ACA CCG CCA GCC TCTTAT TCC ATG TAC AGC CAC 927 Tyr Ser Gly Leu Ser Thr Pro Pro Ala Ser TyrSer Met Tyr Ser His 185 190 195 200 CTC CCT GTC AGC AGC CTC CTC TTC TCCGAT GAG GAG TTT CGG GAT GTC 975 Leu Pro Val Ser Ser Leu Leu Phe Ser AspGlu Glu Phe Arg Asp Val 205 210 215 CGG ATG CCT GTG GCC AAC CCC TTC CCCAAG GAG CGG GCA CTC CCA TGT 1023 Arg Met Pro Val Ala Asn Pro Phe Pro LysGlu Arg Ala Leu Pro Cys 220 225 230 GAT AGT GCC AGG CCA GTC CCT GGT GAGTAC AGC CGG CCG ACT TTG GAG 1071 Asp Ser Ala Arg Pro Val Pro Gly Glu TyrSer Arg Pro Thr Leu Glu 235 240 245 GTG TCC CCC AAT GTG TGC CAC AGC AATATC TAT TCA CCC AAG GAA ACA 1119 Val Ser Pro Asn Val Cys His Ser Asn IleTyr Ser Pro Lys Glu Thr 250 255 260 ATC CCA GAA GAG GCA CGA AGT GAT ATGCAC TAC AGT GTG GCT GAG GGC 1167 Ile Pro Glu Glu Ala Arg Ser Asp Met HisTyr Ser Val Ala Glu Gly 265 270 275 280 CTC AAA CCT GCT GCC CCC TCA GCCCGA AAT GCC CCC TAC TTC CCT TGT 1215 Leu Lys Pro Ala Ala Pro Ser Ala ArgAsn Ala Pro Tyr Phe Pro Cys 285 290 295 GAC AAG GCC AGC AAA GAA GAA GAGAGA CCC TCC TCG GAA GAT GAG ATT 1263 Asp Lys Ala Ser Lys Glu Glu Glu ArgPro Ser Ser Glu Asp Glu Ile 300 305 310 GCC CTG CAT TTC GAG CCC CCC AATGCA CCC CTG AAC CGG AAG GGT CTG 1311 Ala Leu His Phe Glu Pro Pro Asn AlaPro Leu Asn Arg Lys Gly Leu 315 320 325 GTT AGT CCA CAG AGC CCC CAG AAATCT GAC TGC CAG CCC AAC TCG CCC 1359 Val Ser Pro Gln Ser Pro Gln Lys SerAsp Cys Gln Pro Asn Ser Pro 330 335 340 ACA GAG GCC TGC AGC AGT AAG AATGCC TGC ATC CTC CAG GGT TCT GGC 1407 Thr Glu Ala Cys Ser Ser Lys Asn AlaCys Ile Leu Gln Gly Ser Gly 345 350 355 360 TCC CCT CCA GCC AAG AGC CCCACT GAC CCC AAA GCC TGC AGC TGG AAG 1455 Ser Pro Pro Ala Lys Ser Pro ThrAsp Pro Lys Ala Cys Ser Trp Lys 365 370 375 AAA TAC AAG TTC ATC GTG CTCAAC AGC CTC AAC CAG AAT GCC AAA CCA 1503 Lys Tyr Lys Phe Ile Val Leu AsnSer Leu Asn Gln Asn Ala Lys Pro 380 385 390 GGG GGG CCT GAG CAG GCT GAGCTG GGC CGC CTT TCC CCA CGA GCC TAC 1551 Gly Gly Pro Glu Gln Ala Glu LeuGly Arg Leu Ser Pro Arg Ala Tyr 395 400 405 ACG GCC CCA CCT GCC TGC CAGCCA CCC ATG GAG CCT GAG AAC CTT GAC 1599 Thr Ala Pro Pro Ala Cys Gln ProPro Met Glu Pro Glu Asn Leu Asp 410 415 420 CTC CAG TCC CCA ACC AAG CTGAGT GCC AGC GGG GAG GAC TCC ACC ATC 1647 Leu Gln Ser Pro Thr Lys Leu SerAla Ser Gly Glu Asp Ser Thr Ile 425 430 435 440 CCA CAA GCC AGC CGG CTCAAT AAC ATC GTT AAC AGG TCC ATG ACG GGC 1695 Pro Gln Ala Ser Arg Leu AsnAsn Ile Val Asn Arg Ser Met Thr Gly 445 450 455 TCT CCC CGC AGC AGC AGCGAG AGC CAC TCA CCA CTC TAC ATG CAC CCC 1743 Ser Pro Arg Ser Ser Ser GluSer His Ser Pro Leu Tyr Met His Pro 460 465 470 CCG AAG TGC ACG TCC TGCGGC TCT CAG TCC CCA CAG CAT GCA GAG ATG 1791 Pro Lys Cys Thr Ser Cys GlySer Gln Ser Pro Gln His Ala Glu Met 475 480 485 TGC CTC CAC ACC GCT GGCCCC ACG TTC GCT GAG GAG ATG GGA GAG ACC 1839 Cys Leu His Thr Ala Gly ProThr Phe Ala Glu Glu Met Gly Glu Thr 490 495 500 CAG TCT GAG TAC TCA GATTCT AGC TGT GAG AAC GGG GCC TTC TTC TGC 1887 Gln Ser Glu Tyr Ser Asp SerSer Cys Glu Asn Gly Ala Phe Phe Cys 505 510 515 520 AAT GAG TGT GAC TGCCGC TTC TCT GAG GAG GCC TCA CTC AAG AGG CAC 1935 Asn Glu Cys Asp Cys ArgPhe Ser Glu Glu Ala Ser Leu Lys Arg His 525 530 535 ACG CTG CAG ACC CACAGT GAC AAA CCC TAC AAG TGT GAC CGC TGC CAG 1983 Thr Leu Gln Thr His SerAsp Lys Pro Tyr Lys Cys Asp Arg Cys Gln 540 545 550 GCC TCC TTC CGC TACAAG GGC AAC CTC GCC AGC CAC AAG ACC GTC CAT 2031 Ala Ser Phe Arg Tyr LysGly Asn Leu Ala Ser His Lys Thr Val His 555 560 565 ACC GGT GAG AAA CCCTAT CGT TGC AAC ATC TGT GGG GCC CAG TTC AAC 2079 Thr Gly Glu Lys Pro TyrArg Cys Asn Ile Cys Gly Ala Gln Phe Asn 570 575 580 CGG CCA GCC AAC CTGAAA ACC CAC ACT CGA ATT CAC TCT GGA GAG AAG 2127 Arg Pro Ala Asn Leu LysThr His Thr Arg Ile His Ser Gly Glu Lys 585 590 595 600 CCC TAC AAA TGCGAA ACC TGC GGA GCC AGA TTT GTA CAG GTG GCC CAC 2175 Pro Tyr Lys Cys GluThr Cys Gly Ala Arg Phe Val Gln Val Ala His 605 610 615 CTC CGT GCC CATGTG CTT ATC CAC ACT GGT GAG AAG CCC TAT CCC TGT 2223 Leu Arg Ala His ValLeu Ile His Thr Gly Glu Lys Pro Tyr Pro Cys 620 625 630 GAA ATC TGT GGCACC CGT TTC CGG CAC CTT CAG ACT CTG AAG AGC CAC 2271 Glu Ile Cys Gly ThrArg Phe Arg His Leu Gln Thr Leu Lys Ser His 635 640 645 CTG CGA ATC CACACA GGA GAG AAA CCT TAC CAT TGT GAG AAG TGT AAC 2319 Leu Arg Ile His ThrGly Glu Lys Pro Tyr His Cys Glu Lys Cys Asn 650 655 660 CTG CAT TTC CGTCAC AAA AGC CAG CTG CGA CTT CAC TTG CGC CAG AAG 2367 Leu His Phe Arg HisLys Ser Gln Leu Arg Leu His Leu Arg Gln Lys 665 670 675 680 CAT GGC GCCATC ACC AAC ACC AAG GTG CAA TAC CGC GTG TCA GCC ACT 2415 His Gly Ala IleThr Asn Thr Lys Val Gln Tyr Arg Val Ser Ala Thr 685 690 695 GAC CTG CCTCCG GAG CTC CCC AAA GCC TGC TGAAGCATGG AGTGTTGATG 2465 Asp Leu Pro ProGlu Leu Pro Lys Ala Cys 700 705 CTTTCGTCTC CAGCCCCTTC TCAGAATCTACCCAAAGGAT ACTGTAACAC TTTACAAT 2525 TCATCCCATG ATGTAGTGCC TCTTTCATCCACTAGTGCAA ATCATAGCTG GGGGTTGG 2585 GTGGTGGGGG TCGGGGCCTG GGGGACTGGGAGCCGCAGCA GCTCCCCCTC CCCCACTG 2645 ATAAAACATT AAGAAAATCA TATTGCTTCTTCTCCTATGT GNNNNNNNNN NNNNNNNN 2705 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 2765 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 2825 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 2885 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 2945 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 3005 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 3065 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 3125 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 3185 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNN 3245 NTTTAAGTAT TGCATCTGTA TAAGTAAGAAAATATTTTGT CTAAAATGCC TCAGTGTA 3305 TGTATTTTTT TGCAAGTGGG GGGTTACAATTTACCCAGTG TGTATTAAAA AAAACCCA 3365 GAACCCAAAA ATCTCCAGAA GGAAAAATGTGTAATTTTGT TCTAGTTTTC AGTTTGTA 3425 TACCCGTACA ACGTGTCCTC ACGGTGCCTTTTTTCACGGA AGTTTTCAAT GATGGGCG 3485 CGTGCACCAT CCCTTTTTGA AGTGTAGGCAGACACAGGGA CTTGAAGTTG TTACTAAC 3545 AACTCTCTTT GGGAATGTTT GTCTCATCCCANTCTGCGTC ATGCTTGTGT GATAACTA 3605 CCGGAGACAG GGTTTGGCTG TGTCTAAACTGCATTACCGC GTTGTAAAAA ATAGCTGT 3665 CAATATAAGA ATAAAATGTT GGAAAGTCGCAAAAAAAAAA AAAAAAAAAA AAAAA 3720 706 amino acids amino acid linearprotein not provided 2 Met Ala Ser Pro Ala Asp Ser Cys Ile Gln Phe ThrArg His Ala Arg 1 5 10 15 Asp Val Leu Leu Asn Leu Asn Arg Leu Arg SerArg Asp Ile Leu Thr 20 25 30 Asp Val Val Ile Val Val Ser Arg Glu Gln PheArg Ala His Lys Thr 35 40 45 Val Leu Met Ala Trp Arg Gly Leu Phe Tyr SerIle Phe Thr Asp Gln 50 55 60 Leu Lys Cys Asn Leu Ser Val Ile Asn Leu AspPro Glu Ile Asn Pro 65 70 75 80 Glu Gly Phe Cys Ile Leu Leu Asp Phe MetTyr Thr Ser Arg Leu Asn 85 90 95 Leu Arg Glu Gly Asn Ile Met Ala Val MetAla Thr Ala Met Tyr Leu 100 105 110 Gln Met Glu His Val Val Asp Thr CysArg Lys Phe Ile Lys Ala Ser 115 120 125 Glu Ala Glu Met Val Ser Ala IleLys Pro Pro Arg Glu Glu Phe Leu 130 135 140 Asn Ser Arg Met Leu Met ProGln Asp Ile Met Ala Tyr Arg Gly Arg 145 150 155 160 Glu Val Val Glu AsnAsn Leu Pro Leu Arg Ser Ala Pro Gly Cys Glu 165 170 175 Ser Arg Ala PheAla Pro Ser Leu Tyr Ser Gly Leu Ser Thr Pro Pro 180 185 190 Ala Ser TyrSer Met Tyr Ser His Leu Pro Val Ser Ser Leu Leu Phe 195 200 205 Ser AspGlu Glu Phe Arg Asp Val Arg Met Pro Val Ala Asn Pro Phe 210 215 220 ProLys Glu Arg Ala Leu Pro Cys Asp Ser Ala Arg Pro Val Pro Gly 225 230 235240 Glu Tyr Ser Arg Pro Thr Leu Glu Val Ser Pro Asn Val Cys His Ser 245250 255 Asn Ile Tyr Ser Pro Lys Glu Thr Ile Pro Glu Glu Ala Arg Ser Asp260 265 270 Met His Tyr Ser Val Ala Glu Gly Leu Lys Pro Ala Ala Pro SerAla 275 280 285 Arg Asn Ala Pro Tyr Phe Pro Cys Asp Lys Ala Ser Lys GluGlu Glu 290 295 300 Arg Pro Ser Ser Glu Asp Glu Ile Ala Leu His Phe GluPro Pro Asn 305 310 315 320 Ala Pro Leu Asn Arg Lys Gly Leu Val Ser ProGln Ser Pro Gln Lys 325 330 335 Ser Asp Cys Gln Pro Asn Ser Pro Thr GluAla Cys Ser Ser Lys Asn 340 345 350 Ala Cys Ile Leu Gln Gly Ser Gly SerPro Pro Ala Lys Ser Pro Thr 355 360 365 Asp Pro Lys Ala Cys Ser Trp LysLys Tyr Lys Phe Ile Val Leu Asn 370 375 380 Ser Leu Asn Gln Asn Ala LysPro Gly Gly Pro Glu Gln Ala Glu Leu 385 390 395 400 Gly Arg Leu Ser ProArg Ala Tyr Thr Ala Pro Pro Ala Cys Gln Pro 405 410 415 Pro Met Glu ProGlu Asn Leu Asp Leu Gln Ser Pro Thr Lys Leu Ser 420 425 430 Ala Ser GlyGlu Asp Ser Thr Ile Pro Gln Ala Ser Arg Leu Asn Asn 435 440 445 Ile ValAsn Arg Ser Met Thr Gly Ser Pro Arg Ser Ser Ser Glu Ser 450 455 460 HisSer Pro Leu Tyr Met His Pro Pro Lys Cys Thr Ser Cys Gly Ser 465 470 475480 Gln Ser Pro Gln His Ala Glu Met Cys Leu His Thr Ala Gly Pro Thr 485490 495 Phe Ala Glu Glu Met Gly Glu Thr Gln Ser Glu Tyr Ser Asp Ser Ser500 505 510 Cys Glu Asn Gly Ala Phe Phe Cys Asn Glu Cys Asp Cys Arg PheSer 515 520 525 Glu Glu Ala Ser Leu Lys Arg His Thr Leu Gln Thr His SerAsp Lys 530 535 540 Pro Tyr Lys Cys Asp Arg Cys Gln Ala Ser Phe Arg TyrLys Gly Asn 545 550 555 560 Leu Ala Ser His Lys Thr Val His Thr Gly GluLys Pro Tyr Arg Cys 565 570 575 Asn Ile Cys Gly Ala Gln Phe Asn Arg ProAla Asn Leu Lys Thr His 580 585 590 Thr Arg Ile His Ser Gly Glu Lys ProTyr Lys Cys Glu Thr Cys Gly 595 600 605 Ala Arg Phe Val Gln Val Ala HisLeu Arg Ala His Val Leu Ile His 610 615 620 Thr Gly Glu Lys Pro Tyr ProCys Glu Ile Cys Gly Thr Arg Phe Arg 625 630 635 640 His Leu Gln Thr LeuLys Ser His Leu Arg Ile His Thr Gly Glu Lys 645 650 655 Pro Tyr His CysGlu Lys Cys Asn Leu His Phe Arg His Lys Ser Gln 660 665 670 Leu Arg LeuHis Leu Arg Gln Lys His Gly Ala Ile Thr Asn Thr Lys 675 680 685 Val GlnTyr Arg Val Ser Ala Thr Asp Leu Pro Pro Glu Leu Pro Lys 690 695 700 AlaCys 705 103 amino acids amino acid single linear peptide not provided 3Asp Gly Ser Phe Val Gln His Ser Val Arg Val Leu Gln Glu Leu Asn 1 5 1015 Lys Gln Arg Glu Lys Gly Gln Tyr Cys Asp Ala Thr Leu Asp Val Gly 20 2530 Gly Leu Val Phe Lys Ala His Trp Ser Val Leu Ala Cys Cys Ser His 35 4045 Phe Phe Gln Ser Leu Tyr Gly Asp Gly Ser Gly Gly Ser Val Val Leu 50 5560 Pro Ala Gly Phe Ala Glu Ile Phe Gly Leu Leu Leu Asp Phe Phe Tyr 65 7075 80 Thr Gly His Leu Ala Leu Thr Ser Gly Asn Arg Asp Gln Val Leu Leu 8590 95 Ala Ala Arg Glu Leu Arg Val 100 107 amino acids amino acid singlelinear peptide not provided 4 Met Asp Thr Ala Ser His Ser Leu Val LeuLeu Gln Gln Leu Asn Met 1 5 10 15 Gln Arg Glu Phe Gly Phe Leu Cys AspCys Thr Val Ala Ile Gly Asp 20 25 30 Val Tyr Phe Lys Ala His Arg Ala ValLeu Ala Ala Phe Ser Asn Tyr 35 40 45 Phe Lys Met Ile Phe Ile His Gln ThrSer Glu Cys Ile Lys Ile Gln 50 55 60 Pro Thr Asp Ile Gln Pro Asp Ile PheSer Tyr Leu Leu His Ile Met 65 70 75 80 Tyr Thr Gly Lys Gly Pro Lys GlnIle Val Asp His Ser Arg Leu Glu 85 90 95 Glu Gly Ile Arg Phe Leu His AlaAsp Tyr Leu 100 105 106 amino acids amino acid single linear peptide notprovided 5 Met Asn Asn Ser Ser Glu Leu Ile Ala Val Ile Asn Gly Phe ArgAsn 1 5 10 15 Ser Gly Arg Phe Cys Asp Ile Ser Ile Val Ile Asn Asp GluArg Ile 20 25 30 Asn Ala His Lys Leu Ile Leu Ser Gly Ala Ser Glu Tyr PheSer Ile 35 40 45 Leu Phe Ser Asn Asn Phe Ile Asp Ser Asn Glu Tyr Glu ValAsn Leu 50 55 60 Ser His Leu Asp Tyr Gln Ser Val Asn Asp Leu Ile Asp TyrIle Tyr 65 70 75 80 Gly Ile Pro Leu Ser Leu Thr Asn Asp Asn Val Lys TyrIle Leu Ser 85 90 95 Thr Ala Asp Phe Leu Gln Ile Gly Ser Ala 100 105 108amino acids amino acid single linear peptide not provided 6 Cys Leu ArgTrp Asn Asn His Gln Ser Asn Leu Leu Ser Val Phe Asp 1 5 10 15 Gln LeuLeu His Ala Glu Thr Phe Thr Asp Val Thr Leu Ala Val Glu 20 25 30 Gly GlnHis Leu Lys Ala His Lys Asn Val Leu Ser Ala Cys Ser Pro 35 40 45 Tyr PheAsn Thr Leu Phe Val Ser His Pro Glu Lys His Pro Ile Val 50 55 60 Ile LeuLys Asp Val Pro Tyr Ser Asp Met Lys Ser Leu Leu Asp Phe 65 70 75 80 MetTyr Arg Gly Glu Val Ser Val Asp Gln Glu Arg Leu Thr Ala Phe 85 90 95 LeuArg Val Ala Glu Ser Leu Arg Ile Lys Gly Leu 100 105 109 amino acidsamino acid single linear peptide not provided 7 Gln Tyr Ser Asn Glu GlnHis Thr Ala Arg Ser Phe Asp Ala Met Asn 1 5 10 15 Glu Met Arg Lys GlnLys Gln Leu Cys Asp Val Ile Leu Val Ala Asp 20 25 30 Asp Val Glu Ile HisAla His Arg Met Val Leu Ala Ser Cys Ser Pro 35 40 45 Tyr Phe Tyr Ala MetPhe Thr Ser Phe Glu Glu Ser Arg Gln Ala Arg 50 55 60 Ile Thr Leu Gln SerVal Asp Ala Arg Ala Leu Glu Leu Leu Ile Asp 65 70 75 80 Tyr Val Tyr ThrAla Thr Val Glu Val Asn Glu Asp Asn Val Gln Val 85 90 95 Leu Leu Thr AlaAla Asn Leu Leu Gln Leu Thr Asp Val 100 105 105 amino acids amino acidsingle linear peptide not provided 8 Gln Leu Gln Asn Pro Ser His Pro ThrGly Leu Leu Cys Lys Ala Asn 1 5 10 15 Gln Met Arg Leu Ala Gly Thr LeuCys Asp Val Val Ile Met Val Asp 20 25 30 Ser Gln Glu Phe His Ala His ArgThr Val Leu Ala Cys Thr Ser Lys 35 40 45 Met Phe Glu Ile Leu Phe His ArgAsn Ser Gln His Tyr Thr Leu Asp 50 55 60 Phe Leu Ser Pro Lys Thr Phe GlnGln Ile Leu Glu Tyr Ala Tyr Thr 65 70 75 80 Ala Thr Leu Gln Ala Lys AlaGlu Asp Leu Asp Asp Leu Leu Tyr Ala 85 90 95 Ala Glu Ile Leu Glu Ile GluTyr Leu 100 105 110 amino acids amino acid single linear peptide notprovided 9 Cys Leu Gln Phe Thr Arg His Ala Ser Asp Val Leu Leu Asn LeuAsn 1 5 10 15 Arg Leu Arg Ser Arg Asp Ile Leu Thr Asp Val Val Ile ValVal Ser 20 25 30 Arg Glu Gln Phe Arg Ala His Lys Thr Val Leu Met Ala CysSer Gly 35 40 45 Leu Phe Tyr Ser Ile Phe Thr Asp Gln Leu Lys Cys Asn LeuSer Val 50 55 60 Ile Asn Leu Asp Pro Glu Ile Asn Pro Glu Gly Phe Cys IleLeu Leu 65 70 75 80 Asp Phe Met Tyr Thr Ser Arg Leu Asn Leu Arg Glu GlyAsn Ile Met 85 90 95 Ala Val Met Ala Thr Ala Met Tyr Leu Gln Met Glu HisVal 100 105 110

What is claimed is:
 1. An isolated, vertebrate nucleic acid molecule ofbcl-6 locus.
 2. A DNA molecule of claim 1 .
 3. A cDNA molecule of claim2 .
 4. A genomic DNA molecule of claim 2 .
 5. An RNA molecule of claim
 1. 6. A human nucleic acid molecule of claim 1 .
 7. A nucleic acidmolecule comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of the nucleic acid molecule of the bcl-6 locus.
 8. A DNAmolecule of claim 7 .
 9. An RNA molecule of claim 7 .
 10. An isolated,vertebrate nucleic acid molecule of claim 3 operatively linked to apromoter of RNA transcription.
 11. A vector which comprises the nucleicacid molecule of claims 2 or
 10. 12. A vector of claim 11 , wherein theisolated nucleic acid molecule is linked to a plasmid.
 13. The nucleicacid molecule of claim 12 designated pGB31 (ATCC Accession No. 75476).14. The nucleic acid molecule of claim 12 designated pGB3s (ATCCAccession No. 75477).
 15. A host vector system for the production of apolypeptide encoded by bcl-6 locus, which comprises the vector of claim11 in a suitable host.
 16. A host vector system of claim 15 , whereinthe suitable host is a bacterial cell, insect cell, or animal cell. 17.A method of producing a polypeptide encoded by bcl-6 locus, whichcomprises growing the host vector system of claim 11 under suitableconditions permitting production of the polypeptide and recovering thepolypeptide so produced.
 18. A polypeptide encoded by the isolated,vertebrate nucleic acid molecule of claim 1 .
 19. An antibody capable ofbinding to polypeptide encoded by bcl-6.
 20. A monoclonal antibody ofclaim 19 .
 21. A polyclonal antibody of claim 19 .
 22. The isolatednucleic acid molecule of claim 1 that is labelled with a detectablemarker.
 23. The isolated nucleic acid molecule of claim 22 , wherein themarker is a radioactive label, a calorimetric, luminescent, or afluorescent marker.
 24. An antagonist capable of blocking the expressionof claim 18 .
 25. The antagonist of claim 24 , wherein the antagonist isa triplex oligonucleotide capable of hybridizing to nucleic acidmolecule of claim 1 .
 26. An antisense molecule capable of hybridizingto the nucleic acid molecule of claim 1 .
 27. The antisense molecule ofclaim 26 , wherein the molecule is a DNA.
 28. The antisense molecule ofclaim 26 , wherein the molecule is a RNA.
 29. A triplex oligonucleotidecapable of hybridizing with a double stranded DNA molecule of claim 2 .30. A transgenic nonhuman mammal which comprises the isolated nucleicacid molecule of claim 1 introduced into the mammal at an embryonicstage.
 31. An assay for non-Hodgkin's lymphoma, comprising (a)incubating a sample of suitable body fluid for a subject with amonoclonal antibody reactive with non-Hodgkin's lymphoma cells to asolid support, (b) removing unbound body fluid from the support, and (c)determining the level of antigen activity exhibited by the bound bodyfluid to the support.
 32. A method for screening putative therapeuticagents for treatment of non-Hodgkin's lymphoma, which comprisesdetermining in a first sample from a subject with non-Hodgkin's lymphomathe presence of the isolated nucleic acid molecule of claim 1 ,administering to the subject a therapeutic amount of the agent such thatthe agent is contacted with the cell associated with the condition,determining after a suitable period the amount of the isolated nucleicacid molecule in a sample from the treated subject, and comparing theamount of isolated nucleic acid molecule determined in the first samplewith the amount determined in the sample from the treated subject, adifference indicating the effectiveness of the agent, thereby screeningputative therapeutic agents for treatment of non-Hodgkin's lymphoma. 33.A method for diagnosing B-cell lymphoma in a subject comprising: (a)obtaining DNA sample from the subject; (b) cleave the DNA sample intofragments; (c) separating the DNA fragments by size fractionation; (d)hybridizing the DNA fragments with a nucleic acid molecule comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of the nucleicacid molecule of the bcl-6 locus to detect the DNA fragment containingthe bcl-6 sequence; and (e) comparing the detected DNA fragment from (d)with the DNA fragment from a known normal subject, the difference insize of the fragments indicating the occurrence of B-cell lymphoma inthe subject.
 34. A method of claim 33 , where in step (b), the DNAsample is cleaved by restriction enzyme.
 35. A method of claim 33 ,wherein the size fractionation is step (c) is effected by apolyacrylamide or agarose gel.
 36. A method of claim 33 , where in step(d), the nucleic acid molecule is labeled with a detectable marker. 37.A method of claim 36 , wherein the detectable marker is a radiolabelledmolecule, a fluorescent molecule, an enzyme, or a ligand.
 38. A methodof claim 33 , further comprising transferring the DNA fragments into asolid matrix before step (d).
 39. A method for diagnosing B-celllymphoma in a subject comprising: (a) obtaining RNA sample from thesubject; (b) separating the RNA sample into different species by sizefractionation; (c) hybridizing the RNA species with a nucleic acidmolecule comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of the nucleic acid molecule of the bcl-6 locus to detect theRNA species containing the bcl-6 sequence; and (d) comparing thedetected RNA species from step (c) with the RNA species from a knownnormal subject, the difference in size of the species indicating theoccurrence of B-cell lymphoma in the subject.
 40. A method of claim 39 ,wherein the size fractionation in step (b) is effected by apolyacrylamide or agarose gel.
 41. A method of claim 39 , where in step(c), the nucleic acid molecule is labeled with a detectable marker. 42.A method of claim 41 , wherein the detectable marker is a radiolabelledmolecule, a fluorescent molecule, an enzyme, or a ligand.
 43. A methodof claim 39 , further comprising transferring the RNA species into asolid matrix before step (c).
 44. A method of treating a subject withnon-Hodgkin's lymphoma, comprising administering an effective amount ofthe antisense molecule of claim 26 operatively linked to a suitableregulatory element coupled with a therapeutic DNA into a tumor cell of asubject, thereby treating the subject with non-Hodgkin's lymphoma.
 45. Amethod of treating a subject with non-Hodgkin's lymphoma, comprisingadministering an effective amount of the antagonist of claim 23 , and asuitable acceptable carrier, thereby treating the subject withnon-Hodgkin's lymphoma.